Genome-Wide SNP Genotyping in Grape – What is Next?

Part of National Genetic Trait Index ProjectCRIS# 1907-21000-030-00D

USDA-ARSGeneva, Cornell, Davis, Cold Spring

Harbor

Acknowledgement: Sean Miles/Doreen Ware

Team

• Edward Buckler and Sean Myles – Genomics and statistical analysis

• Doreen Ware, Jer-Ming Chia, Bonnie Hurwitz – Bioinformatics

• Charles Simon, Gan-Yuan Zhong, Mallikarjuna Aradhya, Bernard Prins – Germplasm

Genus Vitis• Contains over 60 species mostly found in

temperate regions of the northern hemisphere (Both Old and New World Distributions) – ~3500 accessions

• Vitis vinifera is the most important domesticated species cultivated for table grapes and wine making (~1300 Accessions)

• The wild grape Vitis sylvestris is considered the progenitor of the domesticated grape

• Highly heterozygous and low LD (~200bp)

Genetic Diversity in the Domesticated Grape

Cluster Density Cluster Size

Berry Size Berry Shape?Genetic Diversity

Objectives

1. Grape as a Model Crop for National Genetic Trait Index (NGTI)2. Characterization of Molecular Diversity – Functional Variability3. Genome-wide Association Mapping4. Identify Markers Associated With Economic Traits5. Develop Strategies for Marker Assisted Breeding – Juvenile Selection in Perennial/Tree Crops

Steps• Step 1: SNP Discovery - Next-Generation

Sequencing to sample diversity– DNA preparation, sequencing method and

analysis of sequencing reads for variation– Characterization of SNPs: position, allele

support, and coverage– 10k SNP array development

• Step 2: Genotype and Assemble Data for Analysis

• Step 3: Phenotyping

Step 1: Discovery of genetic variants (SNPs)

Diverse Samples10 cultivated Vitis varieties (Vitis vinifera)

6 wild Vitis species

Genome complexity reduction

Digestion with HpaII restriction enzyme

Illuminia/Solexa sequencingSequencing by synthesis

60 million sequencesTotal: 2 billion base pairs of sequence

Discovery of >1 million SNPs

Make data availableIntegrate SNP data into public

grape genome browser

SNP Discovery Panel

• Goal: Capture recent variation in the genus Vitis

• RRLs constructed from 10 domesticated cultivars and 6 wild species

1. Ehrenfelser2. French Colombard3. Gewurztraminer4. Kadarka5. Malvasia6. Muscat of Alexandria7. Pinot Noir8. Plavac Mali9. Thompson Seedless10. White Riesling

11. Vitis amurensis12. Vitis cinerea13. Vitis labrusca14. Vitis palmata15. Vitis rotundifolia16. Vitis sylvestris

17. Inbred Pinot Noir (Reference Genome)

Library Construction ProtocolReducing the complexity of the Genome

DNA ExtractionDNA Extraction

Whole Genome Amplification*Whole Genome Amplification*

Genome Complexity Reduction: Restriction enzyme digest

Genome Complexity Reduction: Restriction enzyme digest

Size Selection from Gel: 100-600bp

Size Selection from Gel: 100-600bp

Addition of ‘A’ Base to 3`endsAddition of ‘A’ Base to 3`ends

Ligation of Solexa AdaptorsLigation of Solexa Adaptors

Solexa Genome AnalyzerSolexa Genome Analyzer

Next-Generation Sequence Analysis Workflow

Aln Consensus &

Quality

Variation Discovery

Filters

Called SNPs

Variation

Variation Discovery

Gapped Alignment

Ungapped Alignment

Alignments

Mapped to genome?

YES

NO

Read Mapping

Data Accessibility

Image files from

Solexa GA

Base Calling

Sequence and Base Quality

Data Storage

Sequence and Base Quality

Firecrest, Bustard

Deciphering Genetic Diversity From High-Throughput Sequencing

Overview of the Solexa SNP pipeline1. 56 Million reads (1.8 billion bp) are aligned to the

reference genome– The divergence within V. vinifera and with other Vitis is

so great we need to develop other algorithms to map the reads

2. 1.1 Million regions of the genome have potential SNPs, which are statistically evaluated for genotypic basis.

3. 50,000 high probability SNPs are identified4. Empirically validating a small subset of the data.5. With improved algorithms and increased

knowledge of grape diversity, we may be able to extract 100,000s of SNPs.

Mapping Statistics of reads from each of the germplasm to the reference vitis genome

10K SNPs Consequence within Genomic Sequence

• SNP consequence data facilitated via the integration of SNP calls with the genome annotation through Ensembl

• Selected 10K SNPs enriched for genic SNPs.

• In contrast, genome is 46% in genic space, 41% repetitive/transposable elements

10K SNPs: Segregation Patterns

Step 2: Genotyping the grape germplasm repository

SNP selectionChoose 10,000 high quality SNPs

from the 500,000 Solexa SNPs

10K SNP chipProduction of custom 10,000 (8898) SNP genotyping array

Genotype the germplasm repository-1200 cultivated species (Vitis vinifera)

- 1000 wild species

21 million genotypes

Analyses-Establish core germplasm collection-Identify synonyms and homonyms

- Association mapping- Estimate population genetic parameters

PCA analysis of array scored SNPs show clustering of the different germplasm

PCA are able to discriminate between the wild variety

1809 samples 6907 SNPs

Eurasian wild Vitis

American wild Vitis

Differentiation between rotundifolia subspecies

MDS plot Vitis vinifera & Vitis sylvestris 6907 SNPs

MDS plot Vitis vinifera 6907 SNPs

Error or biologically interesting?

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Profiling anthocyanins (525 nm) and other phenolics in grapes (HPLC-DAD chromatograms)

525nm 365nm 280nm

Phenotyping Economic Traits/ Key Secondary Metabolites of Grapes

Phenotyping the USDA-ARS Vitis collections will be the next critical step for maximizing the value of the current genotyping effort

A pilot project has been initiated for phenotyping key secondary metabolites of the Vitis collections from both Davis, CA and Geneva, NY

About 400 V. vinifera and 200 North American collections will be phenotyped for 50 various phenolics including anthocyanins

Funding: CRIS:1907-21000-030-00D

Thanks