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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