Post on 05-Aug-2020
This project is funded by Interna5onal Development Research Centre (IDRC), www.idrc.ca, and the Canadian Government, through Canadian Interna5onal Development Agency (CIDA), www.acdi-‐cida.gc.ca
Genetic diversity and population structure of diploid potato genotypes
The 17th Joint Mee-ng of EAPR Breeding and Varietal Assessment Hévíz, Hungary, June 30 –July 4, 2013
Deissy Juyó Rojas, María F. Álvarez, Andrés Cortés, Helena Brochero, Christiane Gebhardt and Teresa Mosquera
SELECTION
-‐ Without dormancy -‐ Early matura9on -‐ Adapta9on to higher temperatures
S. phureja
Photo: Rodríguez. (2009)
S. stenotomum
(Hawkes, 1987)
2
3
Ghislain et al., 1999; Ghislain et al., 2006; Spooner et al., 2010
The Colombo-‐Ecuatorian mountains are a diversity center for this species
1. (RAPD) -‐ Rela5vely homogeneous group 2. (SSR) -‐ Differen5ated level of ploidy
Potato Breeding Program has been working since 1995.
To improve quality tuber for storage and marke5ng, increasing yield and to look for resistance to pathogens and pest.
Criolla Paisa
Criolla La9na
4
Photos: Rodríguez et al., 2009
• Shape of corolla, s5gma and tuber color. • Precocity. • Industrial processing: significant differences in dry ma\er content, specific gravity and absorp5on of oils. • Resistance to pests (Tecia solanivora) and diseases (Phytophthora infestans)
Escallón and Ramírez, 1997; Mar5n et al., 2000; Ocampo, 2003; Manrique and Ñustez, 2010
Photo: Angarita 2010
To determine the gene5c diversity and popula5on structure of diploid potato genotypes through gene5c characteriza5on with SSR markers.
7
Popula9ons ID Genotypes Origen
Natural
Colombian Core Collec9on CCC 97 Collected in different regions of
Colombia
Gatersleben Bank IPK 13 Different countries
Photo: L. E. Rodríguez
Selec9on and op9miza9on of SSR markers
Molecular characteriza9on • Allele frequency • Polymorphism
Gene9c diversity
Popula9on structure
Popula9on assignment Intrapopula9on Interpopula9on
9
Selection and optimization of SSR markers
Molecular characterization • Allele frequency• Polymorphism
Genetic diversity Population structure
• Population assignmentWithin population
Among population
0%
20%
40%
60%
80%
100%
IPK-17010
IPK-262291
IPK-170021
IPK-17003
IPK-2622011
IPK-262261
IPK-170111
IPK-262321
IPK-17008
IPK-14951
IPK-17009
CCC140
CCC81
IPK-262271
IPK-21495
CCC80
CCC144
CCC70
CCC141
CCC93
CCC143
CCC142
CCC118
CCC103
CCC108
CCC137
CCC145
CCC115
CCC41
CCC120
CCC7
CCC63
CCC62
CCC98
CCC35
CCC123
CCC37
CCC69
CCC133
CCC52
CCC135
CCC136
CCC96
CCC44
CCC51
CCC74
CCC138
CCC8
CCC53
CCC43
CCC114
CCC40
CCC67
CCC116
CCC83
Milbourne et al., 1998, Feingold et al., 2005, Ghislain et al., 2006 -‐ 2009.
Criteria: • Distribu5on in the 12 linkage groups of potato
• Absence of gene5c linkage (> 5 cM)
• Op5miza5on based on the concentra5on of MgCl2, Ta (annealing temperature)
• Op5mal level of resolu5on
Range of distribu5on 1 – 7 SSRs by linkage group
Distance > 10 cM
Ta ± 1-‐ 2 °C [MgCl2] 1 – 3 mM
SSR within the expected size (bp)
Selec9on and op9miza9on of SSR markers
Molecular characteriza9on • Allele frequency • Polymorphism
Gene9c diversity
Popula9on structure
• Popula9on assignment Intrapopula9on Interpopula9on
• Genotyping • Number of alleles/ SSR • Analysis of SSR polymorphic (PIC)
• 23 SSR selected • Silver stain
13
• 19 SSR selected
• Sepreadex gel (EL300, EL600)
SSRs -‐ Poliacrylamide gel 4% SSRs Sepreadex gel STI031
STI022
STGBSS
STM0030
Col- UN MP Col- UN M3
Col- UN M3Col- UN MP
STI031
STI022
STGBSS
STM0030
Col- UN MP Col- UN M3
Col- UN M3Col- UN MP
14
* Sokware Powermarker v.3.25
Number of alleles
Polymorphic index content (PIC) *
SSRs (range) 2 – 12 0.20 – 0.801
Mean 6 0,458
Total per popula9on 240
Selec9on and op9miza9on of SSR markers
Molecular characteriza9on • Allele frequency • Polymorphism
Gene9c diversity
Popula9on structure
• Popula9on assignment Intrapopula9on Interpopula9on
16
Gene9c diversity
Intrapopula9on
Interpopula9on
1. Allelic richness 2. Allelic variants 5. Hardy – Weinberg equilibrium 6. Linkage disequilibrium 7. Heterozigosity
1. Gene5c differen5a5on (F) 2. RST 3. Gene5c flow
17
Allelic variants CCC
(N=97) IPK
(N=13)
Number of alleles 1 – 11 (STI023)
1 – 9 (STI021)
Allelic size (bp) 96 -‐ 270 96 -‐ 310
Rare alleles 32 alleles/22 SSR 1 allele / 1 SSR
Private alleles 37 alleles /21 SSR 24 alleles / 16 SSR
Average frequency of null alleles * 0,1152 0,1905
*Maximum likelihood es5ma5on of null allele frequency (Genepop 4.0.10) EM algorithm (Dempster, Laird and Rubin, 1977) – dis5nguishes between apparent homozygous and heterozygous for different alleles.
18
P-‐ value (locus/popula5ons) Total P-‐ value (popula5ons)
H0= random union of gametes H1= heterozygote excess
Dememoriza5on number = 1000 Number of batches = 100 Number of itera5ons per batch = 1000
Marker CCC FIS
StwaX2 -‐0.344*
STM0025 -‐0.298*
STI054 -‐0.959*
Marker CCC IPK Total test Global
P = 1.000 SD = 0.000
P = 1.000 SD = 0.000
19
Popula9ons CCC IPK
Pairs of markers in LD 64 (7%) P = 0.0000-‐0,0009
6 (0,66%) P = 0,0000-‐0,00013
Common markers in LD (i.e STGBSS, StwaX2, STG0018, STM1104, STM0025)
P-‐value* : Analyzed a\er Bonferroni correc9on for mul9ple comparisons problems (P < 0,0012)
Test associa5on between diploid genotypes at two loci
Ho: "Genotypes at one locus are independent from genotypes at the other locus"
Popula9ons HO HS HT DST GST
Natural 0.424 0.555 0.582 0.026 0.045
20
Ho = Observed heterozygosity Hs = Expected heterozygosity HT = Total heterozygosity
DST = Gene diversity among popula9ons GST = Differen9a9on between popula9ons on several loci
• High heterozygosity (Hs-‐diversity) has been maintained despite the ar5ficial selec5on.
• High intervarietal hybridiza5on (outcrossing condi5ons).
21
Diploid geno9pes from Colombia (0.55)
S. tuberosum group tuberosum (0.46 – 0.52)
S. tubresosum group andigenum (0.88)
Hs
• Diploid potato species have a strong self-‐incompa5bility due to the presence of the S allele rejec5on system, that inhibit fer5liza5on when male and female gametes present the same gene5c cons5tu5on (Hawkes 1987).
A. Gametofi9c B. Sporofi9c
plant S1S2 plant S1S2
S9gma
Style
Ovary
23
Gene9c diversity
Intrapopula9on
Interpopula9on
1. Allelic richness 2. Allelic variants 5. Hardy – Weinberg equilibrium 6. Linkage disequilibrium 7. Heterozigosity
1. Gene5c differen5a5on (F) 2. RST 3. Gene5c flow
24
FIS: Inbreeding coefficient within popula5ons (FIS < 0 indicate heterozygote excess) FST: Coefficient of gene5c differen5a5on between popula5ons FIT: Total inbreeding coefficient
Popula9ons FIS FST FIT Natural 0.17115* 0.09636* 0.25103*
* P-‐ value : 0.00000
25
Population 1 Population 2 Rst Nm**Col IPK 0,23635 0.00000 + 0.0000 * 1,61554Col Spm 0,00888 0.57658 + 0.0364 55,83642Col Cc -0,03417 0.72973 + 0.0497 infinitoCol Spv 0,00157 0.69369 + 0.0490 317,65973IPK Spm 0,23316 0.00000 + 0.0000 * 1,64447IPK Cc 0,09677 0.35135 + 0.0212 4,66704IPK Spv 0,14344 0.04505 + 0.0152 * 2,98587Spm Cc -0,01636 0.52252 + 0.0572 infinitoSpm Spv 0,04732 0.39640 + 0.0454 10,06548Cc Spv 0,01328 0.32432 + 0.0411 37,14959
* Significance values (P ≤ 0,05)** Genetic flow (Nm=M) calculate from Rst
P- valor
M= Nm = Gene9c flow Es5ma5ng migra5on rates
Popula9on 1 Popula9on 2 RST Nm**
CCC IPK 0.23635* 1.61554
Selec9on and op9miza9on of SSR markers
Molecular characteriza9on • Allele frequency • Polymorphism
Gene9c diversity
Popula9on structure
• Popula9on assignment Intrapopula9on Interpopula9on
27
-‐ 42 SSR -‐ Use Admixture Model -‐ Fst = 0.01 and 0.096 -‐ Number of MCMC Reps aker Burning: 300,000 -‐ Length of Burning Period: 30,000 -‐ Number of subgroups—K—ranging from two to ten with ten independent repeats
• The results of Structure showed a con9nuous increase of the goodness of fit, Ln[P(D)], versus the number of groups K
• No popula9on structure was found between ini9al popula9on
• The Δ sta5s5cal test using the STRUCTURE-‐SUM program showed that K = 2 was op5mal in this analysis
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-9400
-9200
-9000
-8800
-8600
-8400
-8200
-8000
-78000 2 4 6 8
Log
. lik
elih
ood
K
Mean [LnP(D)]
Likelihood [LnP(D)]
0
20
40
60
80
100
120
140
160
180
0 1 2 3 4 5 6 7 8
∆K
K
∆K plot (Evanno et al., 2005)
29
IPK CCC
STRUCTURE solu5on. Bar plot of individual potato cul5vars generated by STRUCTURE 2.3.2.1 using the admixture model with independent allele frequencies. Groups are represented by colours, as indicated in the legend. Each column (110 in total) represents a cul5var its genotype and is par55oned into segments indica5ng its likely gene5c origin
0%
20%
40%
60%
80%
100%
IPK-17010
IPK-262291
IPK-170021
IPK-17003
IPK-2622011
IPK-262261
IPK-170111
IPK-262321
IPK-17008
IPK-14951
IPK-17009
CCC140
CCC81
IPK-262271
IPK-21495
CCC80
CCC144
CCC70
CCC141
CCC93
CCC143
CCC142
CCC118
CCC103
CCC108
CCC137
CCC145
CCC115
CCC41
CCC120
CCC7
CCC63
CCC62
CCC98
CCC35
CCC123
CCC37
CCC69
CCC133
CCC52
CCC135
CCC136
CCC96
CCC44
CCC51
CCC74
CCC138
CCC8
CCC53
CCC43
CCC114
CCC40
CCC67
CCC116
CCC83
0%
20%
40%
60%
80%
100%
CCC66
CCC76
CCC119
CCC101
CCC128
CCC129
CCC86
CCC122
CCC34
CCC61
CCC73
CCC30
CCC110
CCC102
CCC4
CCC42
CCC31
CCC131
CCC13
CCC99
CCC132
CCC33
CCC109
CCC21
CCC38
CCC88
CCC27
CCC65
CCC112
CCC124
CCC23
CCC100
CCC106
CCC126
CCC17
CCC104
CCC6
CCC16
CCC117
CCC127
CCC9
CCC14
CCC19
CCC20
CCC71
CCC121
CCC125
CCC11
CCC15
CCC56
CCC79
CCC89
CCC2
CCC3
CCC24
The group of selected SSRs allowed the gene5c characteriza5on of the popula5on of interest. Our results indicated that the popula5on of diploids genotypes from germplasm bank of Colombia has high gene5c diversity, possibly due to the high gene flow and the differen5a5on among individuals within popula5ons. The lack of popula5on structure (CCC) found in this study allows to use this popula5on for gene5c associa5on studies.
30
Trait (QTL) Marker
Associa5on
1. Associa5on mapping
2. Design new strategies to collect genotypes to increase gene5c diversity in the collec5on
31
This project is funded by Interna5onal Development Research Centre (IDRC), www.idrc.ca, and the Canadian Government, through Canadian Interna5onal Development Agency (CIDA), www.acdi-‐cida.gc.ca
Max Planck Ins5tute for Plant Breeding Research Chris9ane Gebhardt
Dr. Klaus J. Dehmer
-‐ Dr. Agim Ballvora -‐ University of Bonn -‐ Álvaro Soler (Interna5onal Center of Tropical Agriculture – CIAT) -‐ David Cuéllar -‐ Universidad Nacional de Colombia
Sponsors
División de Inves5gación Bogotá Universidad Nacional de Colombia
Contract RGE0069 to access gene5c resources from: Ministerio del Ambiente y Desarrollo Sostenible
This project is funded by Interna5onal Development Research Centre (IDRC), www.idrc.ca, and the Canadian Government, through Canadian Interna5onal Development Agency (CIDA), www.acdi-‐cida.gc.ca
Thanks for your a\en5on