community progress toward an integrated toolbox and consensus map for oat nick tinker, eric jackson,...

Community progress toward an integrated toolbox and

consensus map for oat

Nick Tinker, Eric Jackson, Eric Jellen, Howard Rines, et al

Summary

• What do we need (covered in previous talks)• What do we have (quick re-cap)• DArT markers: what and why ?

• New applications• Put in context of yesterday morning• Next steps - and the future

– (Jellen, Jackson, Rines et al)

Marker types – advantages & use in oats

Restriction + Hybridization: RFLP

Random amplification: RAPD

Restriction + amplification: AFLP

Amplification of repeats: SSR

Repeat-based amplification: IRAP, ISSR, REMAP, IMP

Amplification + hybridization: DArT

Targeted amplification: SRAP, SCAR

Targeted – many methods: SNPOther Protein,

morphology

MASDiscovery Oats

Inventory of oat maps (hexaploid)

• Kanota x Ogle (O’Donoughue et al. 1995, Wight et al. 2003)• Kanota x Marion (Kianian et al. 1999, Groh et al. 2000)• Clintland64 x IL86-5698 (Jin et al. 2000)• Ogle x TAM 0-301 (Portyanko et al. 2001) • Ogle x MAM17-5 (Zhu and Kaeppler 2003)• Terra x Marion (De Koeyer et al. 2004)• MN841801 x Noble (Portyanko et al. 2005)• + + + Many other ‘special purpose’ partial maps • + + + Additional new maps, in progress…

– Potoroo x Mortlock (Williams et al.) – Aslak x Matilda (Schulman et al.)– Buffalo x Tardis (Howarth et al.)– SolFi x HiFi (Rossnagel et al.)

Inventory of oat QTLs

• Based on survey of literature (Wight):– 710 reported QTL in oat– From 49 different oat populations– 43 different traits – 81 ‘trait methods’

• http:// avena.agr.gc.ca/oatgenes

6[0

] bcd

1867

[20]

umn136

[29]

isu1755a

[42]

isu1146a

[56]

bcd1872b

[65]

cdo1313

[68]

pgd

[73]

umn815b

[78]

cdo82

[87]

pgi

[94]

isu1372a

3+38[0] cdo1449[7] og43

[23] umn433[27] bcd1405[33] bcd454[36] wg719b

[62] bcd1150[67] cdo590b[73] aco221b[75] cdo395b[81] cdo1319a

[87] aco124[92] bcd1660[100] cdo1527a[109] aco226b

[134] bcd1532a[136] cdo549a[142] bcd907[151] umn107a

[158] umn133a

3831

[0] cdo393c

[15] cdo346b

[24] wacgcac123

2[0] aco193crv

[5] cdo1466

[12] bcd1851c

[16] cdo1158

[24] bcd1829a

[28] cdo1342

[36] cdo539a

[39] umn5846

4_1

2

[0] a

vnA

[11] b

cd1482b

[18] cd

o1423a

[26] cd

o718b

[34] cd

o580

[40] u

mn5301_3

c

[46] cd

o1173a

[53] re

4m

2_7

x

[81] p

ta71a

[91] cd

o187

412

1[0] cdo393c

[15] cdo346b

[24] wacgcac123

2[0

] ac

o193

crv

[5]

cdo1

466

[12]

bcd

1851

c

[16]

cdo

1158

[24]

bcd

1829

a

[28]

cdo

1342

[36]

cdo

539a

[39]

um

n584

6 6[0] bcd1867

[20] umn136

[29] isu1755a[42] isu1146a

[56] bcd1872b

[65] cdo1313[68] pgd[73] umn815b[78] cdo82[87] pgi[94] isu1372a

4_12[0] avnA

[11] bcd1482b

[18] cdo1423a

[26] cdo718b

[34] cdo580

[40] umn5301_3c

[46] cdo1173a

[53] re4m2_7x

[81] pta71a

[91] cdo187

412

3+38[0] cdo1449[7] og43

[23] umn433[27] bcd1405[33] bcd454[36] wg719b

[62] bcd1150[67] cdo590b[73] aco221b[75] cdo395b[81] cdo1319a[87] aco124[92] bcd1660[100] cdo1527a

[109] aco226b

[134] bcd1532a[136] cdo549a[142] bcd907

[151] umn107a[158] umn133a

383

• Too much of a good thing?

• Some assembly required!

Many markers, many maps, many QTLs

Where’s the problem?

Comparative mapping

ko_5[0] acor254a

[8] umn5047

[12] umn97b

[18] wg605

[35] cdo1396

[44] cdo1165a

[59] cdo393b

[67] cdo1173b

[72] umn5904b

[76] wg282

[84] umn28brv

[91] cdo1433

ogle_tam_4

bcd1150b [0]

cdo1165a [10]

cdo202 [35]

cisu36b [37]

cdo393a [40]

umn214c [41]cisu72b [44]

bcd1495a [46]

plrk10b [52]

kv1.9b [63]

cdo618 [66]

cisu35a [68]

cdo1387 [73]

umn51b [81]

bcd1230a [91]

cdo20 [96]

cdo1081b [102]

What is missing from the oat toolbox ?

• An economical, efficient, and accurate method to – merge information from different maps – generate a ‘master’ consensus map

• Quickly address new information in new germplasm– new crosses, association mapping– breeding applications

• Universal anchor markers – that anyone can use – available as a service– Locus-specific, retain identity – Can be put on future sequence framework

• SSR? SNP? Yes! – but we couldn’t wait• Not a Band-Aid solution

Institutions Contacts

AAFC (Canada) Nick Tinker, Steve Molnar

CDC, U of Sask. Brian Rossnagel, Graham Scoles

USDA + Cornell University Mark Sorrells, Jean-Luc Jannink

Göteborg University, Sweden Olof Olsson

IGER, UK Catherine Howarth, Tim Langdon

NULS, Norway Åsmund Bjørnstad

Svalöf Weibull Stine Tuvesson

UFRGS Brazil Luiz C. Federizzi

University of Illinois Fred Kolb

USDA Aberdeen, Idaho Mike Bonman, Eric Jackson

USDA + Univ. Minnesota Howard Rines, Marty Carson, Deon Stuthman

USDA + Purdue Univ. Joe Anderson, Herb Ohm

Oat DArT “consortium”

+ Andrzej Killian, DArT P/L. Australia

DART – Marker Discovery

Pooled Genomic DNA- From 100 global varieties- Reduced complexity- Random clones

- 3 discovery arrays-6500 spots per array- (20K potential markers)

Genomic SamplesFrom 200 global varieties+ 80 KxO mapping lines

Label, hybridize, wash, and scan

• Differential spot (usually from differential restriction or amplification in complexity reduction step)

• Score these on discovery panel + mapping lines• Make second-generation typing array• Optional: sequence clones

Variety-1 Variety-2 ….. Variety-200

DART – Marker Discovery

“0”“1”“0”

Preliminary results

(before sequencing)

200 varieties + 80 KxO lines

20

00 p

olym

orph

ic c

lone

s

< 2000 unique (1400)

> 2000 in next arrays (2700)

= about 2000 markers

1000 mapped in KxO

Winter Spring

Dendrogram based on DArT

““

Comparison to the old map: (example)

Cluster of markers

New DArT map Wight et al….

ko_DArT_G1

[0] 454714[3] 454204[5] 468309[6] 455151[7] 468816[8] 466372[10] estd[18] bglucanase[22] 465771[23] 452799[23] 453382[24] 466075[25] 463651[33] 468706[34] 453818[34] 452401[35] 466890[37] 463786[41] 466082[42] 468762[43] 397085[45] 451275[49] 388110[52] 467722[54] 463435[54] bcd1968b[58] umn220[62] 412525

[68] 450476[72] bcd808b[76] umn624

[83] 415995[85] 456275

[90] 463998[92] cdo590a[96] cdo795a[99] 464463[100] 468600[105] 451513[106] 456262[107] 454921[108] 454164[112] 451699[113] 451986[113] 392483[114] 454993[116] 413966[117] 464950[119] 391188[125] 451003[131] 414509[136] 397106[136] 390797[139] bcd961

ko_24_26_34

umn5245_3 [0]

isu1874a [6]

estd [13]

bglucanase [24]

bcd1968b [40]umn220 [43]

bcd808b [53]

umn624 [58]

cdo590a [70]

cdo795a [76]

aco139rv [87]cdo353a [90]

re2m2_12x [104]

wacacaa378 [115]

cdo373 [122]

bcd961 [132]

isu1651 [142]

Refining results(and adding value)

2700 DArT clones were sequenced

(thanks: NAMA, Joe Anderson, Purdue)

Assembly of 2700 DArT clone sequences

Singletons + consensus = 1800 unique sequences

Number of sequences

Singletons(1280)

2 x 290= 580

318

192

Number of Contigs

SingletonsX 1280

290

106

Example of SSR and SNPs in DArT contig

SNPsSSR

BLAST similarity of 1800 unique sequences

of 1800 non redundant sequences 800 have a BLAST hit with E-value < 10E-5

< 10E-5

BLAST DArT against rice genome on NCBI:

Clone#1 Clone#2

Oat vs Rice by DArT clone homology

[0] opt-1495[3] opt-16999[4] opt-7690[9] opt-12215[11] opt-9437[14] opt-12247[15] opt-6880[20] opt-8509[22] opt-0764[27] opt-9635[28] opt-3694[29] opt-8883

[52] opt-13594

[63] opt-5549

[71] opt-12279

[88] opt-14838[92] opt-15112

[98] opt-3782[100] opt-6499[103] opt-9820[104] opt-18307[105] opt-17649[110] opt-16610[115] opt-1813[120] opt-3120[122] opt-3486[125] opt-18274[128] opt-13862[130] opt-4879[137] opt-9293[138] opt-7958[140] opt-8987[141] opt-13447[143] opt-4863[145] opt-12073[146] opt-14893[149] opt-4742[150] opt-5506[159] opt-14793[162] opt-1427[169] opt-18310[170] opt-3216[172] opt-8109[174] opt-7477[175] opt-14436[177] opt-3194[178] opt-10803

rice2

bcd269 [0]opt-16849 [3]opt-9873 [4]opt-2121 [5]

opt-16786 [6]opt-11714 [9]

opt-10919 [10]opt-14516 [12]opt-13366 [12]e8m3_13x [13]

cdo772 [18]opt-6787 [18]

opt-14599 [19]umn202 [23]

opt-3486 [25]

kod_33

bcd1882a [0]opt-8509 [8]

opt-14292 [20]opt-10664 [20]opt-15363 [22]opt-3049 [23]

opt-13969 [24]opt-12729 [24]opt-17088 [26]opt-8774 [27]opt-7857 [29]

opt-16202 [30]opt-14264 [32]cdo1090c [35]

opt-6501 [50]opt-10660 [52]

cdo836arv [64]

opt-17524 [77]cdo1464 [78]

opt-13153 [83]opt-1568 [84]

opt-12680 [85]cdo1436c [87]

umn5112_2 [91]opt-0473 [92]opt-6514 [93]

opt-15784 [94]opt-11634 [95]opt-3550 [97]

opt-12485 [98]opt-2998 [102]

umn5254_2a [107]

kod_11_41+20+45

Cost and availability of DArT

• Available to ‘non-consortium’ as soon as paper is accepted (fall 2008 ?)

• Paper will contain detailed resources• Add information to Graingenes • All sequence will be submitted

• DiversityArrays.com• Current cost of 2700-clone genotyping array:

– $AUD 5500 (approx. 5K) – 94 genotypes

The way forward

• Snapshots of new initiatives and opportunities – Not all DArTS

• Integrative mapping and chromosome assignment (Segue to Rick Jellen)

At AAFC

• DArTs will be cornerstone of AAFC ‘team project’ on association mapping– Panel of 350 lines of global origin– Everyone look for your favourite trait– Overlaps (complements) other panels:

• Jean Luc• ERA proposal

• DArTs will integrate targeted projects…….

Candidate genes: VRN1 – VRN3 Nava, Federizzi, Wight, Tinker

22

24

629b 97b201b

1 2 3

62b 272b

F R

F R

23F R

exon

intron

Consensus sequence:

ATGGCCGGKAGGGAYAGGGAYCCGCTGGTKGTYGGCAGGGTTGTGGGGGACGTGCTGGACCCCTTCGTCCGRACCACCAACCTCAGGGTGACCTTCGG

GAACAGGRCCGTGTCCAACGGCTGCGAGCTCAAGCCGTCCATGGTCGCCCAGCAGCCSAGGGTKGAGGTGGGCGGCAATGAGATGAGGACCTTCTACA

CRCTC……………………GTGATGGTAGACCCAGATGCTCCAAGTCCWAGCGAYCCCAACCTTAGRGAGTATCTCCACTG…………

GCTTGGTGACAGATATCCCSGGTACAACTGGKGCGTCGTTCGGGCAGGAGGTGATGTGCTACGAGAGCCCTCGTCCRACCATGGGGATCCACCGCTTC

GTGCTCGTRCTCTTCCAGCAGCTSGGGCGGCAGACGGTGTACGCCCCCGGGTGGCGCCAGAACTTCAACACCAGGGACTTYGCCGAGCTCTACAACCT

CGGCCMGCCYGTYGCCGCCGTCTACTTCAACTGCCAGCGYGAGGCCGGCTCCGGCGGCAGGAGGATGTACAATTGA

629b 97b

AACCTCAGGGTGACCTTCG

TGATGGTAGACCCAGATGCTC

GCAATGAGATGAGGACCTTCT

CAGGAGGTGATGTGCTACGA

GTCTACTTCAACTGCCAGCG

KO 24-26-34

UFRGS8 x 605 Group 6

[0] 454714[3] 454204[5] 468309[6] 455151[7] 468816[8] 466372[10] estd

[18] bglucanase[22] 465771[23] 452799[23] 453382[24] 466075[25] 463651[33] 468706[34] 453818[34] 452401[35] 466890[37] 463786[41] 466082[42] 468762[43] 397085[45] 451275[49] 388110[52] 467722[54] 463435[54] bcd1968b

[58] umn220

[62] 412525

[68] 450476

[72] bcd808b

[76] umn624

[83] 415995[85] 456275

[90] 463998[92] cdo590a

[96] cdo795a[99] 464463[100] 468600

[105] 451513[106] 456262[107] 454921[108] 454164[112] 451699[113] 451986[113] 392483[114] 454993[116] 413966[117] 464950[119] 391188[125] 451003[131] 414509

[136] 397106[136] 390797[139] bcd961

468688a [0]466075a [0]454949b [2]454898b [3]465088a [4]454292a [6]

463435a [15]464848a [17]450476a [19]455563a [21]

464623b [34]453284b [34]454164b [34]

451986a [42]464950b [44]

455189b [58]452593b [58]

5 10

15

20

25

QTL

0

0.1

0.20.3

0.4

0.50.6

AssociationMapping

R2(unadjusted)

Vernalization QTLsIn wheat and barley ?

QTL for un-vernalized days-to-flower

Other current initiatives (posters etc)

• New maps and new traits…. • Various projects in evolution and diversity• Rossnagel et al – DArT breeder friendly• Olof Olsson – Tilling + ESTs *10K available now• Jackson, Anderson et al: improve SSR toolbox

– Then on to SNPs – Both SSRs and SNPs to incorporate DArTs

Two key puzzle pieces (segue to Rick… )

• Ogle x TAM O-301 (136 RILs) – any day now– Expect 1000 mapped markers– 700 common to KxO– Eric Jackson – expanding population by 200 RILs – Plus other maps– Consensus mapping will still be a challenge

• Physical chromosome assignment– Need to expand work by Fox et al.– New strategies ….

Oat Workshop at PAG Genome XVII

• Saturday - January 10, 2009 • PAG - San Diego - www.intl-pag.org• 4:40 pm - 6:50 pm

• Colleagues and staff at AAFC• Charlene Wight• Steve Molnar, Judith Fregeau-Reid• Weikai Yan, Jennifer Mitchel-Fetch• and many others….

• Collaborators in the DArT consortium• Itamar Nava and Luiz C. Federizzi

The Quaker Oats Company , NAMA