NORWEGIAN UNIVERSITY OF LIFE SCIENCES

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2111 2005

Mycotoxin prevention in cereal crops by enhanced host plant resistance

UMB; Åsmund Bjørnstad, Helge Skinnes, Kassa semagn UMN: Jim Anderson, Howard Rines, Brian Steffenson

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NORWEGIAN UNIVERSITY OF LIFE SCIENCES

www.umb.no

The past year

UMB: Proposal for Norwegian Research Council Food research programme 2006-2009 not granted

Resubmitted

In 2006 600’ NOK from Graminor and ca. 450’ from UMB/NRC has kept the spirit and allowed the work (+ other related projects)

UMN: A major event, the Barley Coordinated Agricultural project (CAP) involving 16 partners coordinated by UMN staff

Important markers in wheat shared by Jim Anderson

An international consortium established for DArT markers in oats, UMN and UMB highly involved

The SCAB-USA programme continues

Purchased lab analyses of mycotoxins from UMN

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The Barley CAP: coordinated from UMN

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The Barley CAP

Obj. 1. High-Throughput Marker Development – Develop an integrated 3,000 single nucleotide polymorphism (SNP) - based genetic/physical/expression map of the barley

genome.

Obj. 2. Worldwide Web Access – Develop an integrated web portal and database for efficient access to map, gene expression, and trait data.

Obj. 3. Genes and Traits – Provide access to economically important genes using association genetics and diverse

breeding germplasm.

Obj. 4. Superior Germplasm – Develop elite barley

germplasm using high-throughput marker-assisted selection (MAS).

Obj. 5. Education and Outreach - Educate students, scientists, farmers, processors, and consumers in use of genomics technology for gene discovery and crop

improvement.

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Gene Expression Data

~500,000 spots

22,840 barley genes

Barley1 GeneChip

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The Barley CAP: prospects

Barley is simpler to work with than wheat and oat – a diploid with 1/3 of the genome size (still it is 16 times greater than the rice genome)

Our most important cereal crop The student/teaching component:

Proposed English spoken M.Sc. Programme in Plant Sciences (plant Biotechnology and plant breeding/genetics) from 2007 Proposed campus-Ph.D. programme in Genetics and breeding?

A mutual information/recognition of courses and student exchange would greatly support such a build-up here in UMB

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Progress in wheat mycotoxin resistance

UMN: getting closer to the cloning of the gene from the Chinese source ’Sumai 3’ on chromosome 3BS

Jim’s work on deriving markers from rice is also paying off The 3BS-256 marker is only 0.6 cM from the gene and is diagnostic in every case tested

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Sumai3 (AUT) Sumai3 (CAN) Sumai3 (CHN) Sumai3 (IRN) Sumai3 (JAP) Sumai3 (USA)

Gwm 533

Gwm 493

Scab Marker

Validation

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Fine Mapping of Qfhs.ndsu-3BS

XBARC133 segregation

Population of ~3,200 F₂’s from a cross between one pair of NILs Sumai 3/Stoa RIL 63//MN97448

3,156 individuals analyzed for recombination between Xgwm533 and Xgwm493 382 recombinants identified

recombinants genotyped with additional markers

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0.87, 3BS-3 0.78, 3BS-8 0.75, 3BS-7

0.57, 3BS-9 0.56, 3BS-2

0.33, 3BS-1

0.20, 3BS-5 FL Line

Xsun2 Xgwm533 XBARC133 Xgwm493 XBARC87 XBARC102

XBARC68

XBARC73

Xgwm389 XSTS3B-1.1 XBARC75

XSTS3B-15 XSTS3B-46 XSTS3B-91 XSTS3B-92 XSTS3B-94 XSTS3B-47 XSTS3B-69 XSTS3B-104 XSTS3B-17 XSTS3B-49 XSTS3B-50 XSTS3B-66 XSTS3B-65 XSTS3B-102 XSTS3B-142 XSTS3B-62 XSTS3B-138 XSTS3B-52 XSTS3B-83 XSTS3B-80 XSTS3B-84 XSTS3B-163 XSTS3B-164 XSTS3B-57 XSTS3B-58 XSTS3B-55 XSTS3B-40 XSTS3B-54

1000 Kb AP003219AP002541AP002487AP003233AP002867

AP002882AP003338AP002868AP003046AP002747

0 Kb

500 Kb

0.0 R687 0.3 C161 0.6 C602

5.3 S1442 5.6 C970 S1543

7.0 S10623 C1679

8.4 R87

8.7 C749 V32 G107 L451

9.5 S13048

10.9 R753 C90 cM Marker

Wheat deletion bin map Chromosome 3BS

Rice BAC/PAC contig Chromosome 1

Rice genetic map Chromosome 1

STS markers developed from

wheat EST Genome 46: 817 - 823 (2003)

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1

C A C C C C G G C G C G G A CT C C C CT C GT C A C A G CT C G A C C TC C G CT C C A G C C G C C G G C A C C A C G

61

G C G T CT C G G TCT CA CT C G C C G C C G C CTC G T C AT C C G C C G C G T C G C C G G A G G T A G A G A A G G

121

A G C C CT C CTC G C C G T C G A C C A C G C C G C C G G A C G A G T C CT C A G C C C TATC G G C C G T G G C G G

181

A G A G C G T G A A G G T G CT C A A G G A G G C G G C C A A G A C G A G G A A G G T G C C G G C C G A C G A C GT G C

241

T G G C G G C G C T G G CC A A G A TC A A G A A G G C C A A G CT C G A C A C CT C C G C CTT CTTC G A G A C G C

>>>>>>>>>>>>>>>>>>>> 301

TC G G C G G G A C C G A G T C C C C C G G C A G G A C AT G G A A G C TCAT CTTCA C C G CT C A G G G

BE499148 Wheat pre-anthesis spike cDNA library

OLIG O start len tm seq

LEFT PRIMER 254 20 59.96 CAA G AT CAA G AA G G C C AA G C RIGHT PRIME R 445 20 60.77 AG G TA C A C C C C GTT CT C G AT SE Q U E N C E SIZE: 445

PRODUCT SIZE: 192

XSTS3B-138

Development of STS Markers

AP002868 against wheat EST

EST Score E-value CA48625 1273 0.0

BE401694 317 8e-83 CD912193 274 1e-69 CD905429 266 3e-67 CD902302 266 3e-67 CD881365 266 3e-67 BJ303565 266 3e-67 BI479894 266 3e-67 BE444237 258 6e-65 BQ842359 256 3e-64 AL825571 254 1e-63 BE606515 252 4e-63

………….. …. …...

BE499148 192 3e-45

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cM 0

50

100

150

19.8

LOD

3.0

XBARC73 XBARC101 Xfba91 Xgwm533.2 Xfbb185 XSTS3B-55 XBARC102 XBARC87 Xgwm493 XSTS3B-52 XSTS3B-138 XBARC133 Xgwm533.1 Xsun2 XBARC75 XSTS3B-1.1 XSTS3B-15 Xbcd907z Xgwm389 Xbcd907r

0.0

STS3B-58 STS3B-49 STS3B-66 STS3B-142 STS3B-80

Aver.

Exp. 1 Exp. 2

Interval Mapping of Chromosome 3BS

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Genetic mapping of Fusarium resistance in wheat in UMB (1)

The cross between Arina x NK93604:

¾ NK93604 (Norwegian spring wheat, moderate resistance)

¾ Arina (Swiss winter wheat, moderate resistance)

¾ Published in Genome and Crop Science 2006 The cross between ‘5’ x Avle :

¾ 5 (Sgv/Nobeokabozu/Mm/Sumai3 )

¾ Avle (Norwegian spring wheat, very susceptible) Resistance in oat:

¾ The most severe problems, T-2 and HT-2 toxins

¾ Very little resistance

¾ Very little genomics

¾ Resistance identified in Avena sterilis -derivatives

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The Arina x NK cross: Resistance is availble from both parents and can be combined

0 2 4 6 8 10 12 14 16 18 20

0.2 0.5 0.8 1.1 1.4 1.7 2.0 2.3 2.6

FHB rating

Percentage of DH lines

Arina NK93604 (a)

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The Arina x NK cross: LOW DON levels can also be combined

0 10 20 30 40 50

4.0 12.0 20.0 28.0 36.0 44.0 52.0 >52.0

DON content (ppm)

Percent of DH lines

Both parents (b)

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Resistance in extreme types is very dependable across years

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

20019

20025

20037

20047

20052

20055

20056

20063

20079

20090

20093

20094

20097

20118

DH number F HB v a lu e

^{FHB 2001} ^{FHB 2002}

FHB 2003 MeanFHB

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Linkage map for AxNK93604 (group1)

Black = SSRs, blue = AFLPs, green = DArTs

P36/M60-159 0.0

wPt-5707 2.9

wPt-3983 3.3

wPt-8072 6.3

wPt-9402 6.4

wPt-9423 6.6

P36/M49-258 9.9

Xbarc263 23.3

wPt-2150 24.0

P34/M49-98 26.3

P37/M59-175 30.2

Xwmc336 37.9

P34/M59-129 39.5

Xgwm264 44.4

P34/M49-162 48.9

wPt-3904 54.6

P44/M60-267 56.1

Xbarc119**

66.2

Xgwm666 73.2

Xgwm164**

73.3

Xcfd59**

74.2

Xbarc28**

75.8

Xwmc611*

76.3

wPt-9757*

91.1

Xwmc304 92.2

Xwmc312 93.6

Xcfa2129 95.2

P46/M62-94 107.0

XDuPw38 120.3

P35/M48-141 124.1

P34/M59-277 129.6

Xwmc59 135.2

wPt-5577 140.0

Xbarc213 142.7

Xbarc17 145.8

1A

P46/M50-328**

0.0

Xwmc49 5.3

wPt-8930 8.7

wPt-4107 9.0

wPt-2052 10.1

wPt-2230**

11.2

Xbarc128*

13.2

wPt-3753 18.4

wPt-3889 24.4

wPt-0974 24.6

P44/M48-287 27.3

wPt-7833 29.0

(Xbarc119)**

41.3

wPt-5562 48.1

P46/M59-405 49.0

Xwmc619 55.8

wPt-5678 56.6

P37/M47-178 58.4

(XDuPw532) 65.1

Xwmc611 66.7

P34/M59-192 68.3

Xgwm11 68.7

Xbarc137 69.1

Xbarc187 69.7

(Xbarc120) 70.9

wPt-1374 72.2

P37/M61-173 76.5

XDuPw214.2 77.4

P34/M61-456 81.5

wPt-8168 82.6

wPt-3451 83.5

Xbarc181 83.7

Xgwm274 87.3

XDuPw214.1 87.5

Xbarc188 91.7

Xwmc766 92.1

wPt-1403 92.3

wPt-6975 92.7

P33/M59-474 94.6

P43/M62-400 96.4

wPt-3475 107.3

wPt-0944 107.4

Xwmc44 119.5

Xwmc367 136.9

wPt-6142 wPt-4721 138.7

Xwmc728 139.6

P33/M60-102 140.7

1B

P43/M48-85**

0.0

Xbarc149 17.8

Xwmc147 23.6

Xwmc432 33.5

Xbarc152**

46.4

wPt-3738**

48.7

Xgwm848 66.0

wPt-0413 71.5

wPt-7038 71.6

wPt-9380 71.9

Xgwm191 72.5

Xgwm337 73.4

wPt-5503 74.2

Xcfd59.2 75.3

Xwmc590 75.8

(Xgwm603) 76.9

Xbarc162.2**

78.8

Xbarc162.1 86.5

wPt-8854 90.0

Xwmc36 99.2

Xcfd48.2 107.1

(Xgwm55) 114.7

Xgwm820 118.9

P45/M60-325 123.7

P36/M59-205**

135.2

Xgwm642**

135.8

P45/M60-118**

137.3

1D

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Location of detected QTL for FHB and DON: 1A

1A

0 1 2 3 4 5 6 7

Xwmc312 Xcfa2129

Xwmc59 wPt-5577

Xbarc213

LOD

FHB DON

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Location of detected QTL for FHB: 1B

1B

0 1 2 3 4 5 6

Xbarc188 P43/M

62-400

wPt-3475 Xwmc44

Xwmc367

LOD

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Genetic mapping of Fusarium resistance in wheat in UMB (2) Can other South East Asian sources supplement the dependence on the one from ‘Sumai3’?

A recombinant line from Hungary: ‘5’ (Sgv/Nobeokabozu/Mm/Sumai3 ) Crossed with Avle (Norwegian spring wheat, very susceptible)

¾Markers and field data under way, tested in Ås, Hungary and China

¾Can the NB gene supplement the one from ’Sumai’?

¾Introducing the Sumai 3 gene with marker assisted selection s into Graminor:

• Materials available 2006 for further backcrosses

• Innovation grant from UMB to implement this in Graminor’s breeding scheme

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Fusarium resistance in oat derived from Avena sterlis L.

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The oat as it used to be (1)

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The oat as it used to be (2)

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Why oats?

A minor crop in EU and North America, but a major cereal crop in cool northern areas (1/3 of cereal area in Norway and Finland)

Important rotation crop with wheat and barley

Stress (cold, drought, flooding tolerant), grown in marginal environments

A secondary domesticate: Originated in the Fertile Crescent, but came as a cereal weed to NW Europe which outweeded the original crop

The transformation theory prevailed until the late 1800’s: from a mixed crop of barley and oats only oats were harvested

Along with rye it saved Norway through cold climatic periods in the Iron Age and Viking times

The vikings robbed mycotoxin free wheat collected as tithes in the monasteries

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Mycotoxins in Norwegian oats

In 2004 up to 25000 µg/kg of DON with severe germination problems in certified seed

In 2005 it was difficult to obtain acceptable quality oats for horse feed.

At least 90% can be removed by dehulling

May in reality mean that shrivelled seeds are blown through the combine

Yield losses due infection are unknown

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Incidence (%>20µg/kg=ppb) of mycotoxins in Norwegin grain samples (Langseth & Rundberget, Norwegian Veterinary

Institute.

DON HT2 +T-2

Barley

n= 62 1997 18 31

n= 40 1998 15 8

Oats

n=84 1997 79 71

n=80 1998 30 64

Wheat

n=62 1997 23 5

n=81 1998 11 0

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Infected panicles in the cv. ’Hurdal’ inoculated with F. graminearum: discoloured or empty spikelets

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Infected panicles in the cv. ’Hurdal’ inoculated with F. graminearum: pink mycelium or empty spikelets

with infected rachilla

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Infected panicles in the cv. ’Hurdal’ inoculated with F. graminearum: Degrees of seed discoloration and shrivelling

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Resistance in oat lines from A. sterilis

The secondary domestication has left oats with probably less genetic variation than e.g. barley

K.J. Frey used Avena sterilis in the USA to introgress novel traits into oats (resistance, high oil, high protein, yield etc.) In 1990 we started a project with him to broaden the base of Nordic oat breeding by making a Nordic/North-American/

Avena sterilis gene pool – since subject to recurrent selection for 6 cycles

The Avena sterilis sources had been backcrossed into US cultivars

In 2001 we tested them for resistance to Fusarium culmorum and were surprised:

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Seed infection in oats: left susceptible cultivar ’Belinda’, right resistant ’615-4’

This seed infection test correlates better with mycotoxins (DON) than visual spike symptoms

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The A. sterilis derivatives have much less infection than normal oats

Freezer blotter test for inoculation test in greenhouse with Fusarium culmorum. Results 10 days after freezing.

0 5 10 15 20 25 30 35

Belinda Hurdal D 921-643 Z 615-4 B 605-X Z 595-7

% high + medium infected seeds

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The infection/resistance process in oats

When are oats infected: at anthesis or later?

How can infection be scored?

Which pathogen species prevail?

Is there resistance available?

What are the resistance mechanisms?

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Genetic studies of the resistance in oats

A great interest in these sources

Crosses have been made with ’Hurdal’ and ’Belinda’ and SSD populations are now in the F4 generation, will be tested in 2007/08

More detailed studies of the infection process are needed More detailed studies of yield component effects are needed To be able to handle this by marker-assisted selection, markers FIRST need to be there

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Genetic studies in oats

Orphan cereal- little research and getting less…

Increasing interst as a health-y crop

Not related to the other cereals (tribe Festucoideae, not Triticeae)

Marker systems undeveloped, only a crude map avaiable in a cross developed largely in UMN

Retro-transposon based systems being developed in Finland DArT consortium with UMN, AgCanada and others in

collaboration with DArT PL (Australia) 2006-07 Open source technology

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Conclusions

Quantitative resistance from A. sterilis exists and can be used for oat improvement

The resistance is dependent on infection conditions and need careful experimentation in inoculation and scoring

Detailed studies of the infection process is needed: when can toxins be ’dehulled’?

Genetic/breeding studies are under way – with continued funding…