understanding the genetic landscape of puccinia graminis f.sp. tritici from a global to a country...
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Les Szabo, Botma VisserTRANSCRIPT
ì Understanding the genetic landscape of Puccinia graminis
f. sp. tritici from a global to a country perspective
Les J. Szabo USDA ARS Cereal Disease Laboratory St. Paul, Minnesota, USA
Botma Visser Department Plant Sciences University of Free State Bloemfontein 9300, South Africa
Co-‐Authors:
ChrisIna Cuomo & S. Sakthikumar Broad InsItute MIT and Harvard Cambridge, MA USA
Jerry L. Johnson USDA ARS Cereal Disease Laboratory St. Paul, Minnesota, USA
Robert F. Park ACRCP, Plant Breeding InsItute University of Sydney Eveleigh New South Wales, Australia
Pgt genotyping project
ì ObjecIves: ì Characterize the global geneIc landscape. ì Develop molecular diagnosIc tools. ì Track regional and global movement. ì Elucidate evoluIon of Pgt.
Pgt genotyping project: Partners
Robert Park
Univ. of Syndey
USDA ARS CDL
Zak Pretorius & Botma Visser
Univ. of Free State
Tom Fetch
Agriculture & Agi - FoodSarah Hambleton
Agriculture & Agi - Food
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Silvia German
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Y. Anikster & H. Sela
Tel. Aviv University
Alexey Morgounov
CIMMYT
Funding: DRRW phase II, USDA
Pgt genotyping project: Methods
ì Genome-‐wide genotyping ì Re-‐sequencing
ì Illumina paired-‐end reads (101 bases, 20-‐30X) ì 70 isolates analyzed ì ~ 1 million SNPs
ì Pgt SNP Chip ì 1,536 SNP loci ì 153 isolates analyzed
ì SNP Panel ì Ug99 Race Group (Ug99 RG) ì 25 SNP Loci
DistribuIon of Pgt isolates genotyped
1 to 56 to 10
Number of isolates
11-20 >20
Total number = 189 Pgt isolates
Pgt genetic landscape
ì Genome-‐wide genotyping ì Re-‐sequencing ì Pgt SNP Chip
ì Combined data sets. ì 1,156 SNP loci ì Principle coordinate analysis
Africa Asia Australia Europe North America South America
Coordinate 3
83ETH6_1
ETH2010_01a (TRTTF)
83KEN_7a
Ug99 RG
96ZIM2a 84KEN8c 86MAR185c 2009KEN06b (RTRJP)
87KEN_3016_4 85MAR_42_2 84ETH_19_A 87KEN_11_4 80MAR_33_B 09ETH_8_3 (JRCQC)
AFRICA
Africa Asia Australia Europe North America South America
Ug99 RG
96ZIM2a 84KEN8c 86MAR185c 09KEN06b (RTRJP) 60IRN_10_b
83ETH6_1
87KEN_3016_4 85MAR_42_2 84ETH_19_A 87KEN_11_4 80MAR_33_B 09ETH_8_3 (JRCQC)
ETH2010_01a (TRTTF)
86PAK_1030_a (RRRT) 06YEM34_1 (TRTTF) ISR_2083_1 (TRRTF)
83KEN_7a
ISR_2147 (TTTTC) ISR_2117 (TTJTC) 12TUR_6_c_1
12TUR_3_A_1 12TUR_6_B-‐2
Coordinate 3
AFRICA ASIA
Africa Asia Australia Europe North America South America
AFRICA ASIA AUSTRALIA
Ug99 RG
87KEN_3016_4 85MAR_42_2 84ETH_19_A 87KEN_11_4 80MAR_33_B 09ETH_8_3 (JRCQC)
96ZIM2a 84KEN8c 86MAR185c 09KEN06b (RTRJP) 60IRN_10_b
ISR_2147 (TTTTC) ISR_2117 (TTJTC) 12TUR_6_c_1
83ETH6_1
ETH2010_01a (TRTTF)
86PAK_1030_a (RRRT) 06YEM34_1 (TRTTF) ISR_2083_1 (TRRTF)
12TUR_3_A_1 12TUR_6_B-‐2
AUS_126_6711
83KEN_7a AUS_194_12356 AUS_326_12356 AUS_21_0
Coordinate 3
Africa Asia Australia Europe North America South America
AFRICA ASIA AUSTRALIA EUROPE
87KEN_3016_4 85MAR_42_2 84ETH_19_A 87KEN_11_4 80MAR_33_B 09ETH_8_3 (JRCQC) 84CSK_764_3 (GHBS)
96ZIM2a 84KEN8c 86MAR185c 09KEN06b (RTRJP) 60IRN_10_b
AUS_126_6711
ISR_2147 (TTTTC) ISR_2117 (TTJTC) 12TUR_6_c_1
83ETH6_1
12TUR_3_A_1 12TUR_6_B-‐2
Ug99 RG
ETH2010_01a (TRTTF)
86PAK_1030_a (RRRT) 06YEM34_1 (TRTTF) ISR_2083_1 (TRRTF)
INRA98_PGT0001
Coordinate 3
83KEN_7a AUS_194_12356 AUS_326_12356 AUS_21_0
84CSK_75_9_C
Africa Asia Australia Europe North America South America
AFRICA ASIA AUSTRALIA EUROPE NA
Coordinate 3
87KEN_3016_4 85MAR_42_2 84ETH_19_A 87KEN_11_4 80MAR_33_B 09ETH_8_3 (JRCQC) 84CSK_764_3 (GHBS)
96ZIM2a 84KEN8c 86MAR185c 09KEN06b (RTRJP) 60IRN_10_b
Ug99 RG
ISR_2147 (TTTTC) ISR_2117 (TTJTC) 12TUR_6_c_1
AUS_126_6711
83ETH6_1
ETH2010_01a (TRTTF)
86PAK_1030_a (RRRT) 06YEM34_1 (TRTTF) ISR_2083_1 (TRRTF)
INRA98_PGT0001
12TUR_3_A_1 12TUR_6_B-‐2
83KEN_7a AUS_194_12356 AUS_326_12356 AUS_21_0
84CSK_75_9_C
01TX27_C (TTTT) 01MN84A (TTTT) 08WA130_1
74MN70_5C (TCLK) 76MN1391 (TMLK) 56IN1970_2C (RKRQ) 80MN518_3 (RKRQ)
91IA154_1 (QCCJ) 72MEX1370_C (QFCQ)
59KS19 (MCCF) 75WA973B (MCCD)
Africa Asia Australia Europe North America South America
AFRICA ASIA AUSTRALIA EUROPE NA SA
Coordinate 3
87KEN_3016_4 85MAR_42_2 84ETH_19_A 87KEN_11_4 80MAR_33_B 09ETH_8_3 (JRCQC) 84CSK_764_3 (GHBS)
96ZIM2a 84KEN8c 86MAR185c 09KEN06b (RTRJP) 60IRN_10_b
Ug99 RG
ISR_2147 (TTTTC) ISR_2117 (TTJTC) 12TUR_6_c_1
AUS_126_6711
83ETH6_1
ETH2010_01a (TRTTF)
86PAK_1030_a (RRRT) 06YEM34_1 (TRTTF) ISR_2083_1 (TRRTF)
INRA98_PGT0001
12TUR_3_A_1 12TUR_6_B-‐2
83KEN_7a AUS_194_12356 AUS_326_12356 AUS_21_0
84CSK_75_9_C
59KS19 (MCCF) 75WA973B (MCCD)
91IA154_1 (QCCJ) 72MEX1370_C (QFCQ)
74MN70_5C (TCLK) 76MN1391 (TMLK) 56IN1970_2C (RKRQ) 80MN518_3 (RKRQ)
01TX27_C (TTTT) 01MN84A (TTTT) 08WA130_1
Pgt geneIc landscape: Ug99 RG
ì SNP panel ì Ug99 RG ì 25 SNP loci
Race Sample ID Genotype A003 A005 A007 A010 A012 A021 A022 A031 A013 A014 A017 A020 A023 A026 A030 A001 A004 A006 A011 A016 A018 A024 A029 A033 A034 TTKSK 98UgA_1 AF-‐001aa CC GG GG TT TT TT CT GG CC AA CC CC AA GG AA AA GG TT CC GG GG CC AA GG AA TTKSK 04Ken156/4 AF-‐001ab CC GG GG TG TT TT CT GG CC AA CC CT AC GG AC AA GG TT CC GG GG CC AA GG AA TTKSK 07Ken34_1 AF-‐001ac CC GG GG TT TT TT CT GG CC AA CC CT AA GG AA AA GG TT CC GG GG CC AA GG AA TTKSK 07Ken11_2 AF-‐001ad CC GG GG TT TT TT CT GG CC AA CC CT AC GG AC AA GG TT CC GG GG CC AA GG AA TTKSK 09Tan06_2 AF-‐001af CC GG GG TT TT TT CT GG CC AA TT CT AC GG AC AA GG TT CC GG GG CC AA GG AA
TTKST 06Ken19v3 AF-‐001ba CT GG GA TT TT TT CT GT TT AA CC CT AC GG AC AA GG TT CC GG GG CC AA GG AA
TTKST 07Ken18 AF-‐001bb CC GG GG TT TT TT CT GT TT AA CC CT AC GG AC AA GG TT CC GG GG CC AA GG AA
TTTSK 07Ken24_4 AF-‐001ca CC GA GG TT TT TT CT GG CC AA TT CT AC GG AC AG GG TC CC GG GG CC AA GA AA
TTTSK 09Tan07_1 AF-‐001cc CC GG GG GG TT TT CT GG CC AA CC CT AC GG AC AA GG TT CC GG GG CC AA GG AA
TTTSK 09Tan08_2 AF-001cd CC GG GG GG TT AT CT GG CC AA CC CT AC GG AC AA GG TT CC GG GG CC AA GG AA
TTKSF UvPgt55 AF-‐001da CC GG GG TT TT AA TT GG CC AG CC CC AA GA AA AA GA TT CC GG AG AC AT GG AG
TTKSF 09Zim01_1 AF-‐001db CC GG GG TT TT AA CT GG CC AA CC CC AA GG AA AA GG TT CC GG GG CC AA GG AA
TTKSF UvPgt61.2 AF-‐001dd CC GG GG TT TT AA CT GG CC AA CC CC AA GA AA AA GG TT CC GG GG CC AA GG AA
PTKST UvPgt60 AF-‐001fa CT GG GG TT TT TT CT GT TT AA CC CT AC GG AC AA GG TT CC GG GG CC AA GG AA
TTKSP UvPgt59 AF-‐001ea CC GG GG TT TG AA TT GG CC AA CC CC AA GA AA AA GG TT CT AG AG AC AT GG AA
Pgt genetic landscape: Ug99 RG
KENYA
ETHIOPIA
ZIMBABWE
UGANDA
SOUTH AFRICA
TANZANIA
ERITREA
Pgt race TTKSK Kenya Uganda
1998 2007 2008 20102004 20122009
AF-001aa
AF-001ad
AF-001ac
AF-001ab
AF-001aa
AF-001afAF-001adAF-001acAF-001ab
AF-001da
AF-001cdAF-001ccAF-001ca
AF-001bbAF-001ba
AF-001ea
AF-001ddAF-001db
AF-001fa
GenotypeTTKSK
PTKST
TTTSK
TTKSF
TTKST
TTKSP
Pgt genetic landscape: Summary
ì Ug99 RG is an unique geneIc group.
ì Individual races of Ug99 RG consist of mulIple genotypes.
ì Genotyping results clearly demonstrates that Pgt has spread globally.
1 to 56 to 10
Number of isolates
11-20 >20
Pgt genetic landscape: Summary
ì This represents the first step in characterizing the Pgt global populaIon.
ì It will take parIcipaIon by the global rust community to complete this project.
Robert Park
Univ. of Syndey
USDA ARS CDL
Zak Pretorius & Botma Visser
Univ. of Free State
Tom Fetch
Agriculture & Agi - FoodSarah Hambleton
Agriculture & Agi - Food
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Silvia German
/E/��ZĞƐĞƌĂĐŚ�/ŶƐƟƚƵƚĞ
Y. Anikster & H. Sela
Tel. Aviv University
Alexey Morgounov
CIMMYT
ì South Africa Pgt genotyping partnership
USDA-‐ARS Prof. Szabo
UFS Dr. Visser, Prof. Pretorius
ARC-‐SGI Dr. Terefe
Pgt DNA SNP
technology
CollecIons
Selected collecIons
ì South Africa Introduction
§ South Africa (http://www.indexmundi.com/agriculture/?country=za&commodity=wheat&graph=production)
§ 29th ranked wheat producer in the world § 2013 production reached 1700 MT
§ Annual Pgt surveys started in South Africa in 1980 (Le Roux and Rijkenberg, 1987)
§ Currently conducted by Dr. Terefe at the Small Grain Institute (ARC-‐SGI) in Bethlehem (see poster 89 for 2011 and 2012 data)
§ Relies on phenotyping viable collections on standard differentials
ì South Africa Introduction
Race 81 82,83 84 85 86S 86W 87S 87W 91 92 93 94 95 96 97 98 99 00 01 02 03 04 07 08 09 10
2SA002 1 3 1.7 9 14 22SA004 0.5 20.2 20.2 9 74 50 77 28 13 3 31 1 26 67 39 1 0.82SA006 31.6 2.8 552SA010 2 9.6 3.6 12SA018 12SA020 0.52SA032 4.5 8.2 8 7 12SA033 12SA036 8.5 1 1 10 22SA039 1.52SA043 0.52SA043 1.4 22SA045 31.12SA048 14.3 100 1.4 12SA049 2.52SA051 12SA052 12SA053 0.52SA054 1.52SA055 65 3.3 3.3 4.52SA088 (TTKSF) 35 68.8 81 85 69.7 26.1 38.4 39 782SA099 5 18.8 3 1 0.82SA100 58.2 55 27 15 29 16 1 5 13 5 2 1 0.82SA101 4.5 5.7 1 12SA102 70 54 36 59 37 53 53 12.4 10 12 23.8 1.1 8.9 12 12SA103 18 46 5 60 21 33 3 6 0.8 22SA104 14.1 9.8 8 32SA105 53.2 20.5 21 152SA106 (TTKSP) 2.2 17.9 14 22SA107 (PTKST) 4 12SA108 (TTKSF+)
100 100 100 100 100 100 100 100 100 100 100 100 100 100 0 100 100 100 100 100 100 100 100 100 100 100
Terefe & Pretorius, 2011a , b
Le Roux and Rijkenberg, 1987 Le Roux, 1989 Boshoff et al ., 2000 Komen, 2007
Terefe et al ., 2010
ì South Africa Introduction
§ SSR analyses identified two unrelated Pgt lineages within South Africa (Visser et al., 2011)
§ Lineage 1 – non-‐Ug99 races § Lineage 2 – Ug99 race group members (see poster 88)
§ Why is the South African situation relevant at a global level?
§ Half of the Ug99 race group members occur in South Africa § Possible movement of races from Africa to Australia was suggested (Watson
and de Sousa, 1982)
ì South Africa Hypothesis
§ The genotyping of South African Pgt samples collected during annual surveys will complement, but not replace, the current phenotyping strategy.
Materials and methods
§ Pgt collections § Dr. Tarakegn Terefe (ARC-‐SGI, Bethlehem, SA) § Surveys
§ 2010 – 84 collections (country-‐wide) § 2011 – 56 collections (country-‐wide) § 2012 – 102 collections (Western Cape Province)
§ CTAB DNA extraction (Saghai-‐Maroof et al., 1984) § Isolate genotyping
§ Ug99 vs non-‐Ug99 identification – Stage 1 qPCR § Ug99 race identification – Stage 2 qPCR § Non-‐Ug99 race identification – SSR analysis (PgSUN7, PgSUN20,
PgSUN27, PgSUN30, PgSUN31, PgSUN33, PgSUN42, PgSUN44 (Karaoglu et al., 2013)
Materials and methods
b
d
Ug99 positive
P5111 P9406
P13470 P18022
Ug99 vs non-‐Ug99 identification
Materials and methods
Ug99 negative
b
d
P5111 P9406
P13470 P18022
Ug99 vs non-‐Ug99 identification
Results
Ug99 vs non-‐Ug99 identification
Year Number of isolates Ug99 positive Ug99 negative Failed DNA extraction
2010 84 64 (76%) 19 (23%) 1 (1%)2011 56 36 (64%) 17 (30%) 3 (6%)2012 102 51 (50%) 49 (48%) 2 (2%)
Materials and methods
Ug99 race identification
Ug99 race A003 A005 A007 A010 A012 A021 A022 A031TTKSF C (H) G (H) G (H) T (H) T (H) A (H) T (F) G (H)
C (H) G (H) G (H) T (H) T (H) A (H) T (F) G (H)TTKSP C (H) G (H) G (H) T (H) T (H) A (H) T (F) G (H)
C (H) G (H) G (H) T (H) G (F) A (H) T (F) G (H)PTKST C (H) G (H) G (H) T (H) T (H) T (F) C (H) G (H)
T (F) G (H) G (H) T (H) T (H) T (F) T (F) T (F)TTKSF+ C (H) G (H) G (H) T (H) T (H) A (H) C (H) G (H)
C (H) G (H) G (H) T (H) T (H) A (H) T (F) G (H)
Results
Ug99 race identification
-‐ 2011 survey
A003 A005 A007 A010 A012 A021 A022 A031Phenotype TTKSF CC GG GG TT TT AA TT GG
TTKSP CC GG GG TT TG AA TT GGPTKST CT GG GG TT TT TT CT GTTTKSF+ CC GG GG TT TT AA CT GG
Isolates S004-‐11 CC GG GG TT TT AA TC GG TTKSF+S005-‐11 CC GG GG TT TT AA TC GG TTKSF+S006-‐11 CC GG GG TT TT AA TC GG TTKSF+S009-‐11 CC GG GG TT TT AA TC GG TTKSF+S015-‐11 CC GG GG TT TG AA TT GG TTKSPS017-‐11 CC GG GG TT TT AA TT GG TTKSFS019-‐11 CC GG GG TT TT AA TT GG TTKSFS021-‐11 CC GG GG TT TT AA TT GG TTKSFS027-‐11 CC GG GG TT TT AA TC GG TTKSF+S030-‐11 CC GG GG TT TT AA TC GG TTKSF+S031-‐11 CC GG GG TT TT AA TT GG TTKSFS032-‐11 CC GG GG TT TT AA TC GG TTKSF+S033-‐11 CC GG GG TT TT AA TT GG TTKSFS034-‐11 CC GG GG TT TT AA TT GG TTKSFS035-‐11 CC GG GG TT TT AA TT GG TTKSFS036-‐11 CC GG GG TT TT AA TT GG TTKSFS037-‐11 CC GG GG TT TT AA TC GG TTKSF+S038-‐11 CC GG GG TT TT AA TT GG TTKSFS039-‐11 CC GG GG TT TT AA TT GG TTKSFS041-‐11 CC GG GG TT TT AA TC GG TTKSF+S042-‐11 CC GG GG TT TT AA TT GG TTKSFS044-‐11 CC GG GG TT TT AA TT GG TTKSFS045-‐11 CC GG GG TT TT AA TT GG TTKSFS046-‐11 CC GG GG TT TT AA TC GG TTKSF+S047-‐11 CC GG GG TT TT AA TT GG TTKSFS048-‐11 CC GG GG TT TT AA TT GG TTKSFS049-‐11 CC GG GG TT TT AA TT GG TTKSFS052-‐11 CC GG GG TT TT AA TT GG TTKSFS053-‐11 CC GG GG TT TT AA TT GG TTKSFS054-‐11 CC GG GG TT TT AA TT GG TTKSFS056-‐11 CC GG GG TT TT AA TC GG TTKSF+S057-‐11 CC GG GG TT TT AA TC GG TTKSF+S058-‐11 CC GG GG TT TT AA TC GG TTKSF+S059-‐11 CC GG GG TT TT AA TC GG TTKSF+S061-‐11 CC GG GG TT TT AA TC GG TTKSF+S062-‐11 CC GG GG TT TT AA TC GG TTKSF+
TTKSF TTKSF+
(see poster 45)
Results
2SA107 2SA88
2SA102
2SA88 2SA88/104
Non-‐Ug99 race identification
-‐ 2010 survey
Results
2SA105/88 2SA105 2SA105 2SA88 2SA105 2SA106
Non-‐Ug99 race identification
-‐ 2010 survey
Results
2SA105/104
2SA105/88
2SA105
Non-‐Ug99 race identification
-‐ 2011 survey
Results
Non-‐Ug99 race identification
-‐ 2012 survey
Results
Non-‐Ug99 race identification
-‐ 2012 survey
Results
Race data summary
Race 98 99 00 01 02 03 04 07 08 09 10 10 11 122SA055 65 3.3 3.3 4.52SA088 (TTKSF) 35 68.8 81 85 69.7 26.1 38.4 39 78 53.7 35.9 372SA099 5 18.8 3 1 0.82SA100 1 0.82SA1012SA102 53 12.4 10 12 23.8 1.1 8.9 12 1 1.9 12SA103 33 3 6 0.8 22SA104 14.1 9.8 8 3 1.22SA105 53.2 20.5 21 15 14.6 24.5 392SA106 (TTKSP) 2.2 17.9 14 2 1.2 1.92SA107 (PTKST) 4 12SA108 (TTKSF+) 22 30.1 142SA105 (?) 7.3 5.7 8Unknown 1
100 100 100 100 100 100 100 100 100 100 100 100 100 100
Current genotyping studyKomen, 2007
Terefe et al ., 2010
Terefe & Pretorius, 2011a , b
ì South Africa Discussion
-‐ Genotyping of Pgt collections -‐ Indicated significant genetic variation within the population -‐ Forced changes in the survey protocol -‐ Confirmed the wide distribution of TTKSF+ before it was detected on
phenotype level -‐ Confirmed possible multiple genotypes for a single phenotype (2SA105) -‐ Indicated the presence of an unknown genotype -‐ Help with planning of future surveys -‐ Confirmed the continued need for phenotyping
-‐ Partnership led to the establishment of Pgt genotyping capacity at UFS with the hope of expanding both in the future
Acknowledgements
§ Prof. Les Szabo (USDA-‐ARS, St Paul, USA) for genotyping partnership
§ BGRI for invitation and financial support to report on partnership
§ Dr. Tarakegn Terefe (ARC-‐SGI, Bethlehem, SA) for Pgt collections
§ Mr. Tonny Selinga (UFS) for technical assistance
§ Prof. Liezel Herselman (UFS)
§ Prof. Zakkie Pretorius (UFS)
Literature cited
-‐ Boshoff WHP, van Niekerk BD and Pretorius ZA, 2000. Suid Afrikaanse Tydskrif vir Plant en Grond 17: 60-‐62.
-‐ Karaoglu H, Lee CMY and Park R, 2013. Australasian Plant Pathology 42: 271-‐281. -‐ Komen JS, 2007. Studies on chemical control of wheat stem rust. M.Sc. Dissertation, University of the Free
State, Bloemfontein, South Africa. -‐ Le Roux, 1989. Phytophylactica 21: 255-‐258. -‐ Le Roux J and Rijkenberg, 1987. Phytophylactica 19: 467-‐472. -‐ Saghai-‐Maroof MA, Soliman KM, Jorgensen RA and Allard RW, 1984. Proceedings of the National
Academy of Sciences USA 81: 8014–8018. -‐ Terefe T and Pretorius ZA, 2011a. 47th Congress of the South African Society for Plant Pathology, 23-‐26
January, Kruger National Park, South Africa. -‐ Terefe T and Pretorius ZA, 2011b. BGRI Technical Workshop, 13-‐16 June, St Paul, Minnesota, USA. -‐ Terefe T, Pretorius ZA, Paul I, Mebalo J, Meyer L and Naicker K, 2010. South African Journal of Plant and
Soil 27: 163-‐167. -‐ Visser B, Herselman L, Park RF, Karaoglu H, Bender CM and Pretorius ZA, 2011. Euphytica 179: 119-‐127. -‐ Watson IA and de Sousa CNA, 1982. Proceedings of the Linnaean Society NSW 106: 311-‐321.