International Potato Center (CIP), Lima, Peru

Awais Khanawais.khan@cgiar.org

June 2, 2015

Genomic-Assisted Breeding inSweetpotato: Current status and future

opportunities

New paradigm of Genomics-AssistedBreeding

Varshney et al. 2014

Importance of selection in plant breeding

General steps in plant breeding (modified after Gepts 2002)

Systematic procedure for genetic improvementthrough crossing plants with desired traits andselecting progeny with improved performanceand/or improved combinations of traits.

Phenotypic selection: Selection based onappearance and performance

I. Difficult to separate environmental & genetic contributionII. Difficult to distinguish homozygous & heterozygous effectsIII. Needs large space & labor inputIV. Slow & time consuming

NaturalPopulations

ProgenyTesting

BreedingPopulation

Selected individuals(phenotypic selection)

Elite (high EBV)individuals

Repeat overgenerations

Improvedindividuals

DNA based selection methods

A. Marker-assisted selection: Selectionfor one or more (up to 8-10) alleles

B. Marker-assisted backcrossing: Oneor more (up to 6-8) donor alleles aretransferred to an elite line

C. Genome-wide selection: Selection ofseveral loci using genomic estimatedbreeding values (GEBVs) based ongenome-wide marker profiling

Trends Plant Sci 10: 621 630

Marker genotype

Single marker Analysis

Is there a significant link between geneticmakeup (genotype) and trait phenotype?

QTL analysis

Overview: Association mapping analysis

Zhu et al. 2008

P1 P2

F1

X

F1 X

F2

F2

RILs

More than 6generations ofselfing

Segregating population

The Maize Nested Association MappingPopulation (NAM)

SSD=Singleseed descent

9

Concept of Marker assisted selectionMolecular breeding

Association between molecular marker and causative gene

Direct association Indirect association SNP in LD with gene

Causative geneSNP within gene

Hirschhorn & Daly, 2005

LD

Sequencing of3 Gbase

genome to18X coverage(54 Gbases)

Sanger 454 Illumina

No. of plates:

Time:

Total cost:

Cost/Mbase:

756,000 120 348 years 6 months 2-3 weeks

$108 millions 1 million $60k

$2,000 $18.5 $3

Cost and throughput comparisons

DNA marker technology coupled withNext-Generation Sequencing (NGS)

Barcodes are incorporated into the adaptors that areligated to the DNA after shearing. It enable thepooling of several different samples in one library.

Multiple samples per sequencing run

Predicting the phenotype: Genomic selection

Hayes et al. 2009

Prediction Accuracy of Genomic Selection

Correlation between

1. LD between markers and QTLs ( LD)2. Size of Training population ( n)3. Heritability of the trait in question ( h2)4. Genetic structure of the trait ( #QTLs)

Accuracy of GS

Affected by:

GEBV(Genomic estimated

breeding value)

EBV(Experimentally estimated

breeding value)

• Tropical root crop from the morning glory family(Convolvulaceae)

• Fifth most important crop in developing countries (afterrice, wheat, maize, and cassava) to prevent malnutrition,and enhanc food security

• Perennial, grown as annual, propagated - storage rootsor stem cuttings

• Orange-fleshed sweetpotato - important source of β-carotene (a precursor of vitamin A)

Sweetpotato (Ipomoea batatas)

• Hexaploid species with 2n = 6x = 90

• Autopolyploid – Allopolyploid!

• Self-incompatibility, out-crossing, and highly-heterozygous,within plot variation is extremely high that needs a largenumber of replicates

• Cultivar development - Phenotype-based selection

Sweetpotato: Challenges for genetic studiesand breeding

• 7,783 accessions at CIP’s genebank (including breeding lines,improved varieties, landraces, and wild accessions)

• Great experience with breeders and scientists at CIP, NARsin SSA, NCSU, LSU, China, Japan and Korea but limitedcommunity compared to many other crops

• Few genetic mapping populations and limited genomicresources – So far few linkage maps with SSRs and AFPLmarkers are available (e.g., Ukoskit et al. 1997; Kriegner et al.2003; Cervantes-Flores et al., 2008)

• QTL mapping in Beauregard and Tanzania: Root-knotnematode resistance, beta carotene content, dry matter,starch content, maltose and sucrose content, iron, zinc,calcium content (Cervantes-Flores et al., 2008, 2011, Chang et al. 2009)

Sweetpotato: Genetic and genomic resources

Sweetpotato Gene Index

Sweetpotato Gene Index from Root

A partially complete genetic linkage map (43 and 47 LGS) basedon retrotransposon insertion polymorphisms.Monden et al. 2015, Breeding Science

Transcriptome of two sweetpotato cultivars Xushu 18 andXushu 781. PAG 2014

Sweepotato genes and genome46 SSR marker based kit identifying1029 alleles, 5 to a 23alleles, averaging 11.5 alleles per marker. Rossel et al. 2014

Whole-genome sequencing (de novo) of two lines of I. trifida,using the Illumina HiSeq platform. Assembled genomesequence from 513-712 Mb. 62,407 and 109,449 putativegenes, 1,464,173 SNPs and 16,682 CNVs. Hirakawa et al. 2015

“Transcriptome Analysis and Genome Wide Association Studiesin Sweet Potato (Ipomoea batatas L. Lam)” (2014-2017): USDAfunded project at University of Arkansas at Pine Bluff.http://www.reeis.usda.gov/web/crisprojectpages/1004277-transcriptome-analysis-and-genome-wide-association-studies-in-sweetpotato-ipomoea-batatas-l-lam.html

Next generation sequence-based genotyping forIpomea trifida

~ 3 Million sequence reads in total and ~1.3 are good reads

SNPs without filtering 5466

SNPs after eliminating NN3643

SNPs after eliminating SNPsnot matching betweenreplicates 3210

SNPs that are polymorphicand segregating in themapping population 646

CropTotal “CROP" and"QTL" “CROP" and "QTL" 2014 “CROP" and "QTL" 2000-2014

Wheat 30900

4400: every possible category(seed size and shape, stripe rustresistance, yield, height, etc.)

17,500: protein, fusariumresistance, agronomic traits

Rice 34200 used as model4770: many genes alreadyidentified associated with QTLs

18500: yield, grain size, abioticstress tolerance

Soybean 19000 2750: protein content, biotic stress1500: yield, abiotic and bioticstress tolerance, agronomic traits

Sugarcane 6110609: not many QTLs actually insugrcane 4400: sugar content

Potato 16,300

1750: tuberization, droughtresistance, tuber shape, carotenoidcontent

12,400 results, mostly bioticresistance

Sweetpotato 4810

259: starch content, storage rootyield

2470:yield, starch, bioticresistance, beta carotene

Cassava 2510 327: starch, viral resistance2270:bacterial blight, plantarchitecture, root bulking

Yams 974 92 841

Source: Google scholar May 31, 2015

QTLs for sweetpotato relative to other crops

CropTotal “CROP" and“Genomic"

“CROP" and “Genomic"2014

“CROP" and “Genomic" 2000-2014

Wheat127,000Genome: 2012 13900 20600

Rice131,000Genome: 2002 15700 24700

Soybean64,200Genome: 2010 9780 18700

Sugarcane 21600 3590 16500

Potato67400Genome: 2011 9610 18900

Sweetpotato 17400 1640 11700

Cassava15400Genome: 2012 1780 12200

Yams 6320 653 4450

Source: Google scholar May 31, 2015

Genomics for sweetpotato relative to other crops

CropTotal “CROP" and“Marker"

“CROP" and “Marker"2014

“CROP" and “Marker" 2000-2014

Wheat 167,000 12700 23400

Rice 110,000 9510 45,500

Soybean 72300 8340 19500

Sugarcane 22000 3110 16400

Potato 71,800 8400 18500

Sweetpotato 18,600 1480 12200

Cassava 16,200 1660 12600

Yams 10,600 880 7390

Source: Google scholar May 31, 2015

Markers for sweetpotato relative to other crops

Genome sequence

Next generation molecularmarkers

Dense genetic maps

Exome Capture

What do we need?

Clear Breeding Goals!• General:

– High-yielding lines (storage root)– Taste and nutrition– Resistance to biotic and abiotic stresses– Dry Matter

• Specific: Depending on program and the region, includeo Beta-carotene levelso Mineral (iron, zinc) contento Starch contento Sucrose contento Drought, heat, and salt toleranceo SPVD, weevil toleranceo Biomass traitso Ornamental traits

What do we need?

Phenotypic traits

Qualitative traitQuantitative traitQualitative traitFall into discrete classes, controlled bytwo or many alleles of single gene andless influenced by environment e.g., bloodtype, seed coat color, many diseases

The wrinkled-seedcharacter of pea iscaused by a transposon-like insertion in a geneABO geneABO gene

The quantitative trait hascontinuous variation (bell-shapedcurve, normal distribution) and isusually controlled by many genesof small effect, or by a few genesof large effect e.g., Height,Weight, Biomass, Diseaseresistance

ButA single polymorphic locus withmultiple, differentially expressedalleles can also result incontinuous variation

Parents

F1

X

Quantitative trait

Additive genetic variance (VA): Each allele has aspecific value that it contributes to the finalphenotype

Example

AABB X aabb

AaBb

A=4 U, a=2 U, B=6 U,b=3 U

Additive effectF1= 15 U (4+2+6+3)

Dominant effectF1=20 U (4+4+6+6)

F1

Parents

Dominance genetic variance (VD): Dominant geneaction masks the contribution of the recessivealleles at the locus

VP = VG + VE + VGE

P = phenotypic, G = genetic, E =environmentalVGE = variation associated with thegenetic and environmental interactionsVG (The total genetic variation)VG = VA + VD + VI

A=additive, D=dominance, I= interaction due to epistatis

Variance components

Interaction genetic variance (VI)/epistasis:Due to masking of genotypic effects at one locus bygenotypes of another locus

The total phenotypic variance can be rewritten asVP = VA + VD + VI + VE + VGE

Genotype-Environment interaction (VGE)Due to difference in the direction of performance ofgenotypes in different environmental circumstances

Environmental variance (VE)Due to difference in magnitude of performance ofgenotypes in different environments

Variance components

Broad-sense heritability: Ratio of total genetic variance tototal phenotypic varianceH2 = VG/VP

Narrow-sense heritability: Ratio of additive geneticvariance to total phenotypic varianceh2 = VA/VP Specific to the population and environment

Does not indicate the degree to which a trait isgenetic, it measures the proportion of the phenotypicvariance that is the result of genetic factors

The proportion of the genetic variance to the total variance

Heritability

High-throughput, precise, accurate and standardizedphenotyping in multiple environments

Chlorophyllcontent SPAD

ReflectanceNDVI

Infrared thermographyfor canopy temperature

What do we need?

Analytical tools for trait analysis, rapid identification of markersfrom sequence data marker-trait associations and genomicselections (most of these tools have to be suitable forhexaploid and clonally propagated crops!)

Database to store andaccess genotypic,phenotypic andenvironment data

What do we need?

Thank you for attention

CACACACACACA

CACACACACACACACA

CACACACACACACA

CACACACACACACACA

CACACACACACACA

CACACACACACACACACACACACACA

CACACACACA

CACACACACA

CACACACACACA

CACACACACACA

CACACACACACACA

P1 P2

XF1

(Allele a) (Allele c)

1

2

3

4

(Allele d)(Allele b)

P1 P2 1 2 3 4

ac

ad

bc

bd

ab cd ac ad bc bd

Genotyping-by-Sequencing

Linkage group 1 and 2 ofPapaya: Map was constructedusing microsatellite markersin F2 population (Chen et al.2007)

Linkage group 1 and 2 ofPapaya: Map was constructedusing microsatellite markersin F2 population (Chen et al.2007)

A21188_4310.0EST_2000.3A21506_425A21506_386B5508_674

4.2

E-AGG_M-CTG_0885.0E-ACT_M-CAC_1896.1B42148_1366.6E-ACT_M-CAC_1907.0B13391_18610.9U10046_26212.6A24217_38014.1A44864_71114.3E-AAC_M-CGG_19414.4E-AGG_M-CAC_13914.5E-ACA_M-CAC_218A204_709B6691_828A23431_262B42209_163A34234_64A17376_241A1700_286A23818_1247A17697_137B8670_316B34819_352A31938_1164A21785_454A4163_165A28717_273EST_207E-ACA_M-CGT_176

14.7

aa23431aa21785E-AGG_M-CTG_118A1700_335aa4163A23431_711

14.8

A43712_18015.1A21785_36915.5A21785_37015.7A27143_498A27143_71916.7EST_52717.1idaaLEAFY3A22253_314A23780_514A23780_350

17.6

B25609_65518.1E-AAC_M-CGG_15619.1E-ACT_M-CAC_12719.8E-AGG_M-CAA_13120.3A12288_57221.5E-ACT_M-CAC_48722.1A5495_22523.6U21184_8423.8E-ACT_M-CAC_366E-ACC_M-CAG_325A7830_328E-ACA_M-CTG_351

23.9

A8249_41024.1A11079_851A20359_445A11079_1298aa1106E-ACT_M-CAC_104E-ACA_M-CGG_404

24.3

A12221_24925.4aa36701A27835_40926.7E-ACA_M-CGT_19026.8A22374_26127.9EST_12032.3A23928_13241.0E-ACT_M-CAC_27446.8A44703_26747.9A44703_41048.0A14364_20549.4A11290_49550.1A20972_60853.2A32488_36757.1A35140_52757.6B1086_66057.9EST_33858.5A22347_45065.4A16565_21667.4E-ACC_M-CAA_10668.8U13442_26769.0A21909_451A21530_120A21530_223

69.1

E-AGG_M-CAA_13869.5A13545_15269.9aa2190971.1

LG1

E-AGG_M-CAA_3840.0E-ACC_M-CAC_2283.0E-ACT_M-CAC_2283.2A13451_96710.7E-AAC_M-CCT_45313.8E-ACT_M-CAC_33321.2E-ACC_M-CAG_12322.7A12796_28722.9A4143_38426.2aa2062229.5EST_21929.8A2794_23137.8A17096_90538.5E-ACC_M-CAA_075A25506_48038.9E-ACC_M-CAG_205E-AAC_M-CCT_153E-AAC_M-CTG_213

39.0

A20672_400E-ACA_M-CAA_172E-ACA_M-CGT_135E-AGG_M-CCA_225

39.1

E-AAC_M-CCT_27439.3E-ACC_M-CAC_07839.4E-ACA_M-CTG_29139.5A11122_811A33808_73A43_308

39.6

aa1112239.7E-AAC_M-CGG_21639.8Max339.9EST_22540.1A30540_380EST_97A18263_133A7236_200

40.4

E-ACA_M-CGT_133E-AGG_M-CTG_311E-ACA_M-CAA_277

40.5

E-ACA_M-CAA_262E-ACA_M-CGT_07840.6A45132_171U37658_160E-AAC_M-CGT_146

40.8

E-ACA_M-CAA_111E-AGG_M-CTG_22041.1E-ACC_M-CAC_081U3704_1032U3704_1469U3704_1466U3704_1129B31408_348

41.4

E-AGG_M-CTG_191E-AAC_M-CCA_10941.7A35106_246A3050_43241.8B19699_33942.0U3704_237E-AAC_M-CGT_094A6628_314B41829_103B31408_621

42.1

A1808_25242.2A7876_659U38236_83342.3E-ACA_M-CAA_12046.9A20529_23249.5B42100_35749.6A38913_39252.1E-ACT_M-CAC_22553.2A33330_36768.3EST_54369.4E-ACA_M-CAC_21280.0A37208_22980.2

LG2

aa234130.0A23413_1710.1E-ACA_M-CAC_1180.2A3946_9995.4A13289_18410.6A13289_17810.8E-AAC_M-CGT_06213.9B21852_152518.6B21852_1257U24480_44119.5U24480_548B11117_26022.9U6734_12423.2B28302_9723.4U17904_173125.2aa334325.7B28302_238228.4E-AGG_M-CAA_18829.6A37074_18931.1EST_42232.1EST_83E-AAC_M-CCT_30433.7U25119_16433.9A9935_25834.3A26458_636B3166_13434.6E-AGG_M-CAA_20434.8A12453_56835.6E-ACA_M-CGG_19736.3E-AGG_M-CCA_36737.2A2087_54037.3E-ACC_M-CAG_06337.5E-AAC_M-CCT_06537.8E-AAC_M-CCT_39539.2E-ACA_M-CGG_247A1531_166A19391_174A5143_243A3799_309

39.3

E-AGG_M-CCA_21539.4A1422_388A15645_221A12659_194A29094_245A5071_238A18286_474E-ACC_M-CAC_243E-ACC_M-CAC_206E-AAC_M-CGT_273EST_540E-AGG_M-CAA_280E-AAC_M-CGT_191E-AAC_M-CCA_139aa29094

39.7

E-AAC_M-CCT_064aa1265939.8E-ACA_M-CAA_25039.9B37157_45240.0A43528_88540.1A41905_64840.7A21119_35640.8A8549_42241.3EST_32141.4A9746_14341.6A41905_459A10377_448U15727_915U15727_1121

41.9

E-AGG_M-CAA_24542.1U15727_114842.3B31654_17142.5E-ACA_M-CGG_36844.3A15081_78045.0E-ACA_M-CAA_08249.7U28660_32555.7E-AAC_M-CCT_31764.3E-AAC_M-CGT_08367.8A28035_46982.5E-AGG_M-CTG_23591.1A12306_42491.6A44090_52593.4A5554_85694.3A10569_19094.8aa6285103.0

LG3

A21188_4310.0EST_2000.3A21506_425A21506_386B5508_674

4.2

E-AGG_M-CTG_0885.0E-ACT_M-CAC_1896.1B42148_1366.6E-ACT_M-CAC_1907.0B13391_18610.9U10046_26212.6A24217_38014.1A44864_71114.3E-AAC_M-CGG_19414.4E-AGG_M-CAC_13914.5E-ACA_M-CAC_218A204_709B6691_828A23431_262B42209_163A34234_64A17376_241A1700_286A23818_1247A17697_137B8670_316B34819_352A31938_1164A21785_454A4163_165A28717_273EST_207E-ACA_M-CGT_176

14.7

aa23431aa21785E-AGG_M-CTG_118A1700_335aa4163A23431_711

14.8

A43712_18015.1A21785_36915.5A21785_37015.7A27143_498A27143_71916.7EST_52717.1idaaLEAFY3A22253_314A23780_514A23780_350

17.6

B25609_65518.1E-AAC_M-CGG_15619.1E-ACT_M-CAC_12719.8E-AGG_M-CAA_13120.3A12288_57221.5E-ACT_M-CAC_48722.1A5495_22523.6U21184_8423.8E-ACT_M-CAC_366E-ACC_M-CAG_325A7830_328E-ACA_M-CTG_351

23.9

A8249_41024.1A11079_851A20359_445A11079_1298aa1106E-ACT_M-CAC_104E-ACA_M-CGG_404

24.3

A12221_24925.4aa36701A27835_40926.7E-ACA_M-CGT_19026.8A22374_26127.9EST_12032.3A23928_13241.0E-ACT_M-CAC_27446.8A44703_26747.9A44703_41048.0A14364_20549.4A11290_49550.1A20972_60853.2A32488_36757.1A35140_52757.6B1086_66057.9EST_33858.5A22347_45065.4A16565_21667.4E-ACC_M-CAA_10668.8U13442_26769.0A21909_451A21530_120A21530_223

69.1

E-AGG_M-CAA_13869.5A13545_15269.9aa2190971.1

LG1

E-AGG_M-CAA_3840.0E-ACC_M-CAC_2283.0E-ACT_M-CAC_2283.2A13451_96710.7E-AAC_M-CCT_45313.8E-ACT_M-CAC_33321.2E-ACC_M-CAG_12322.7A12796_28722.9A4143_38426.2aa2062229.5EST_21929.8A2794_23137.8A17096_90538.5E-ACC_M-CAA_075A25506_48038.9E-ACC_M-CAG_205E-AAC_M-CCT_153E-AAC_M-CTG_213

39.0

A20672_400E-ACA_M-CAA_172E-ACA_M-CGT_135E-AGG_M-CCA_225

39.1

E-AAC_M-CCT_27439.3E-ACC_M-CAC_07839.4E-ACA_M-CTG_29139.5A11122_811A33808_73A43_308

39.6

aa1112239.7E-AAC_M-CGG_21639.8Max339.9EST_22540.1A30540_380EST_97A18263_133A7236_200

40.4

E-ACA_M-CGT_133E-AGG_M-CTG_311E-ACA_M-CAA_277

40.5

E-ACA_M-CAA_262E-ACA_M-CGT_07840.6A45132_171U37658_160E-AAC_M-CGT_146

40.8

E-ACA_M-CAA_111E-AGG_M-CTG_22041.1E-ACC_M-CAC_081U3704_1032U3704_1469U3704_1466U3704_1129B31408_348

41.4

E-AGG_M-CTG_191E-AAC_M-CCA_10941.7A35106_246A3050_43241.8B19699_33942.0U3704_237E-AAC_M-CGT_094A6628_314B41829_103B31408_621

42.1

A1808_25242.2A7876_659U38236_83342.3E-ACA_M-CAA_12046.9A20529_23249.5B42100_35749.6A38913_39252.1E-ACT_M-CAC_22553.2A33330_36768.3EST_54369.4E-ACA_M-CAC_21280.0A37208_22980.2

LG2

aa234130.0A23413_1710.1E-ACA_M-CAC_1180.2A3946_9995.4A13289_18410.6A13289_17810.8E-AAC_M-CGT_06213.9B21852_152518.6B21852_1257U24480_44119.5U24480_548B11117_26022.9U6734_12423.2B28302_9723.4U17904_173125.2aa334325.7B28302_238228.4E-AGG_M-CAA_18829.6A37074_18931.1EST_42232.1EST_83E-AAC_M-CCT_30433.7U25119_16433.9A9935_25834.3A26458_636B3166_13434.6E-AGG_M-CAA_20434.8A12453_56835.6E-ACA_M-CGG_19736.3E-AGG_M-CCA_36737.2A2087_54037.3E-ACC_M-CAG_06337.5E-AAC_M-CCT_06537.8E-AAC_M-CCT_39539.2E-ACA_M-CGG_247A1531_166A19391_174A5143_243A3799_309

39.3

E-AGG_M-CCA_21539.4A1422_388A15645_221A12659_194A29094_245A5071_238A18286_474E-ACC_M-CAC_243E-ACC_M-CAC_206E-AAC_M-CGT_273EST_540E-AGG_M-CAA_280E-AAC_M-CGT_191E-AAC_M-CCA_139aa29094

39.7

E-AAC_M-CCT_064aa1265939.8E-ACA_M-CAA_25039.9B37157_45240.0A43528_88540.1A41905_64840.7A21119_35640.8A8549_42241.3EST_32141.4A9746_14341.6A41905_459A10377_448U15727_915U15727_1121

41.9

E-AGG_M-CAA_24542.1U15727_114842.3B31654_17142.5E-ACA_M-CGG_36844.3A15081_78045.0E-ACA_M-CAA_08249.7U28660_32555.7E-AAC_M-CCT_31764.3E-AAC_M-CGT_08367.8A28035_46982.5E-AGG_M-CTG_23591.1A12306_42491.6A44090_52593.4A5554_85694.3A10569_19094.8aa6285103.0

LG3