genetic diversity and population structure of diploid potato … · 2018-06-08 · ccc66 ccc76...

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)



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



Gene9c diversity

Popula9on structure


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



Gene9c diversity

Popula9on structure


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

28

-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


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


Thanks for your a\en5on

genetic diversity and population structure of diploid potato … · 2018-06-08 · ccc66 ccc76...

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