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Comparative analyses of the potato Comparative analyses of the potato and tomato transcriptomesand tomato transcriptomes

David Francis, AllenDavid Francis, AllenVan DeynzeVan Deynze, John Hamilton, , John Hamilton, Walter De Jong, David Douches, Sanwen Huang, Walter De Jong, David Douches, Sanwen Huang, and C. and C. Robin BuellRobin Buell

Supported by the AFRI Plant Breeding, Genetics, and Supported by the AFRI Plant Breeding, Genetics, and Genomics Program of USDA’s National Institute of Food and Genomics Program of USDA’s National Institute of Food and

AgricultureAgriculture

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Questions

International Sol Project: How can a common set of genes/proteins give rise to such a wide range of morphologically and ecologically distinct organisms?

SolCAP: How can variation be harnessed to improve varieties that benefit the consumer, processors, and the environment?

Sequence data available to address these questions: Draft genome for doubled monoploid DM1-3 516R44 (S. tuberosum L. Phureja group); S. tuberosum, S. lycopersicum, S. pimpinellifolium GAII transcriptomes

Technology

Next Generation Sequencing

SNP genotyping

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What comparisons do we want to make?

How well do S. tuberosum expressed sequences align to DM1-3 516R44 genomic sequences?

How well do S. lycopersicum expressed sequences align to DM1-3 516R44 genomic sequences?

How is variation distributed within a Species?

within a market class?

within a variety?

within a gene?

Which sequence variation is important to phenotypic variation?

Library creation/QC GAII sequencing (single and paired end)

Data Collection Assembly

400

300

Analysis:transcriptome complexitySNP calling/validation

identification of genes under selection

Sample Total ClustersTotal PE Reads

PF Passed Clusters

% PF Passed Clusters

Total PE PF Reads

Actual PE Reads

Atlantic 1 7,601,277 15,202,554 6,382,748 83.97 12,765,496

Atlantic 2 10,544,542 21,089,084 9,252,168 87.74 18,504,336 30,185,186

Premier 1 7,812,394 15,624,788 6,652,121 85.15 13,304,242

Premier 2 11,678,379 23,356,758 9,999,926 85.63 19,999,852 31,949,096

Snowden 1 7,996,418 15,992,836 6,837,553 85.51 13,675,106

Snowden 2 11,781,671 23,563,342 10,393,322 88.22 20,786,644 33,288,120

Illumina GA II Output for Potato

Velvet Assemblies of Potato Illumina Sequences

• With a minimum kmer of 31 and a minimum contig length of 150bp:

Variety Total GbTranscriptome

Size (Mb)No.

Contigs N50 (bp)Maximum

Contig (Kb)

Atlantic 1.8 38.4 45215 666 11.2

Premier 1.9  38.2 54917 408 6.6

Snowden 2.0 38.2 58754 358 6.9

Velvet Assemblies of Potato Illumina Sequences

• Atlantic:• 45214 contigs• 32520 align with GMAP(95%id, 50%cov)• 27106 align with GMAP(95%id, 90%cov)

• Premier:• 54917 contigs• 41497 align with GMAP (95%id, 50%cov)• 37297 align with GMAP (95%id, 90%cov)

• Snowden:• 58754 contigs• 44479 align with GMAP (95%id, 50%cov)• 40708 align with GMAP (95%id, 90%cov)

Alignment of S. tuberosum GAII-transcriptome contigs to the PGSC draft genome sequence from DM1-3 516R44:

Tomato Illumina GA II Output

VarietyInsert Size

Read Length Total Reads PF Reads %PF Passed Total PF

FL7600 300 61/47 22,491,304 20,685,342 92.0FL7600 300 60 16,025,976 14,382,577 89.8FL7600 300 60 15,645,164 13,985,875 89.4 49,053,794

NC84173 350 61/61 27,079,946 22,687,626 83.8NC84173 350 60 11,058,431 10,366,811 93.8NC84173 350 60 14,401,240 12,687,134 88.1 52,539,617OH9242 350 61/47 26,960,898 24,874,218 92.3OH9242 350 60 10,316,775 9,671,753 93.8OH9242 350 60 14,676,814 12,879,812 87.8 51,954,487

T5 350 61/47 26,799,944 24,677,302 92.1T5 350 60 16,822,639 14,738,351 87.6T5 350 60 15,726,257 13,744,511 87.4 59,348,840

PI114490 350 61/47 17,721,226 16,422,842 92.7PI114490 350 60 17,115,349 14,902,672 87.1PI114490 350 60 17,890,649 15,248,587 85.2 52,727,224PI212816 350 61/47 17,631,906 16,450,422 93.3PI212816 350 60 18,238,179 15,354,882 84.2PI212816 350 84 21,829,622 18,500,235 84.8 57,699,707

Variety Total GbTranscriptome

Size (Mb)No.

Contigs N50 (bp)Maximum

Contig (Kb)

FL7600 2.82 39.8 59,581 424 12.1

NC84173 2.77 39.2 60,534 496 13.3

OH9242 2.70 39.1 59,051 476 11.6

T5 3.04 40.6 60,031 632 14

PI114490 2.70 41 61,310 690 11.7

PI212816 3.00 41.1 66,118 471 14

Velvet Assemblies of TomatoIllumina Sequences

• With a k-mer length of 31 and a minimum contig length of 150bp:

Sequence quality: Viewing an Atlantic potato contig from the Velvet assembly

FL7600 (93.7 % id; 94.4 % coverage)

Snowden (97.9; 94.7)

Alignment of contigs relative to DM1-3 516R44

Query SNPs Filtered SNPs

Atlantic Asm 224748 150669

Premier Asm 265673 181800

Snowden Asm 258872 166253

Identify intra-varietal SNPs

A/C SNP

Filtered SNP counts

Ref Query d 10 d 20 d 30 d 40 d 50 d 60 d 100

atlantic atlantic 21336 17509 14493 12150 10277 8673 4435

atlantic premier 21789 18050 15084 12477 10584 8919 4620

atlantic snowden 19997 16518 13694 11378 9689 8048 4173

premier atlantic 21117 17096 14106 11785 9790 8222 4228

premier premier 22951 18431 15016 12377 10300 8703 4371

premier snowden 20972 16846 13709 11357 9479 7873 4113

snowden atlantic 20777 16998 13984 11619 9647 8131 4186

snowden premier 22101 17888 14701 12068 10124 8650 4223

snowden snowden 21083 16963 13792 11218 9359 7735 3896

Filtering on SNP quality and 1 SNP/ 150bp window

depth of coverage

No. SNPs

Filtered SNP countsFiltering on SNP quality and 1 SNP/ 150bp window

0

5000

10000

15000

20000

25000

Validationrate

40

45

50

55

60

65

70

75

80

85

90

Genotyping platforms….

Comments on quality control…

Data….

direct comparison of sequence

analysis of SNPs across populations

COS

R-gene

Comparison of two genes on tomato chromosome 9 BAC

COSII

Fresh Market vs Fresh Market        Identities = 573/573 (100%), Gaps = 0/573 (0%)Fresh Market vs Processing        Identities = 569/569 (100%), Gaps = 0/569 (0%)S. lycopersicum vs S. pimpinellifolium        Identities = 339/341 (99%), Gaps = 0/341 (0%)Potato vs Potato        Identities = 606/612 (99%), Gaps = 0/612 (0%)Tomato vs Potato          Identities = 914/948 (96%), Gaps = 6/948 (0%)

DIVERGED SEQUENCE

Fresh Market vs Fresh Market        Identities = 959/959 (100%), Gaps = 0/959 (0%)Fresh Market vs Processing        Identities=1560/1560(100%), Gaps=0/1560 (0%)S. lycopersicum vs S. pimpinellifolium        Identities = 612/613 (99%), Gaps = 0/613 (0%)Tomato vs Potato        Identities = 223/280 (79%), Gaps = 11/280 (3%)Potato vs Potato  Identities = 246/278 (88%), Gaps = 7/278 (2%)

What patterns do we expect to see for genes “under selection”?•Low Variation (fixed)•High Ka/Ks (mutations affect protein, possible diversifying selection)•Mutations (loss of function)•FST (genes that distinguish populations)

All 173 markers (K=6)

89 Coding markers (K=5)

84 Non-coding markers (K=6)

Processing Fresh-market Vintage Landrace

500K burnin/750K MCMC reps, 20 runs for each K from 3 to 8

Population structure: coding vs. non-coding

CA & OH OH

CA OH OH

CN

CN

Distribution of FST for genes

ovate: 0fw2.2: 0sp6: 0.14

ovate: 0.26fw2.2: 0sp6: 0.73

ovate: 0.31fw2.2: 0sp6: 0.47

ovate: 0fw2.2: 0.5sp6: 1

ovate: 0fw2.2: 0.42sp6: 0.74

ovate: 0.14fw2.2: 0.46sp6: 0.05

Marker Chrom SourceNo. of SNPs

LEOH14 unknown EST542533 2 Pathogenesis-related leaf protein LEOH16 5 EST301659 4 drought-induced protein LEOH17 multiple EST551464 5 putative alcohol dehydrogenaseLEOH20 unknown EST327354 2 ultraviolet-B-repressible protein LEOH23 2 EST546919 3 Vesicle-associated membrane LEOH25 9 EST511738 3 gamma hydroxybutyrate LEOH29 unknown EST243853 2 A96510 protein F27F5.25 LEOH31 9 EST583372 10 chlorophyll synthetase G4 LEOH32 9 EST358606 2 acetyl-CoA C-acyltransferase LEOH33 9 EST471439 5 endo-1,3-1,4-beta-D-glucanaseLEOH34 9 EST435427 6 tuberisation-related proteinLEOH35 9 EST549543 8 photosystem II protein W CosOH7 2 TOVBN23 5 putative MYB transcription factorLEVCOH11 1 AJ785180.  2 late embryogenesis (Lea)-like

Examples of highly polymorphic genes within S. lycopersicum

0.304 Associated with introgression0.043 Duplicated genes0.087 Ethylene induced0.174 Pathogen induced0.130 Abiotic Stress induced

Note: I am working on a replacement that compares Ka/Ks for selected tomato and potato genes

Marker Chrom SourceNo. of SNPs

LEOH14 unknown EST542533 2 Pathogenesis-related leaf protein LEOH16 5 EST301659 4 drought-induced protein LEOH17 multiple EST551464 5 putative alcohol dehydrogenaseLEOH20 unknown EST327354 2 ultraviolet-B-repressible protein LEOH23 2 EST546919 3 Vesicle-associated membrane LEOH25 9 EST511738 3 gamma hydroxybutyrate LEOH29 unknown EST243853 2 A96510 protein F27F5.25 LEOH31 9 EST583372 10 chlorophyll synthetase G4 LEOH32 9 EST358606 2 acetyl-CoA C-acyltransferase LEOH33 9 EST471439 5 endo-1,3-1,4-beta-D-glucanaseLEOH34 9 EST435427 6 tuberisation-related proteinLEOH35 9 EST549543 8 photosystem II protein W CosOH7 2 TOVBN23 5 putative MYB transcription factorLEVCOH11 1 AJ785180.  2 late embryogenesis (Lea)-like

Examples of highly polymorphic genes within S. lycopersicum

non-synonymous 0.53synonymous 0.37non-coding 0.09

Note: I am working on a replacement that compares Ka/Ks for selected tomato and potato genes

2 3 11 127 8 9 105 641

2

3

4

5

6

7

8

9

<0.001

<0.0001

Combined

12

1

10

11

r 2 value

Chr

omso

me

P v

alue

>0.05

<0.05

<0.01

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.000.70 0.80 0.90 1.000.00 0.10 0.20 0.30 0.40 0.50 0.60

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r 2 value

6 7 8 9 10 11

10

<0.000111

12

1 2 3 4 5

4

<0.01

5

6

<0.0017

8

9

Fresh Market

Chr

omso

me

1

P v

alue

>0.05

2

<0.053

Processing Fresh Market Vintage Wild

Distribution of PM genes across populations is not random

Visit us at http://solcap.msu.edu/

Tools, Downloads

Conclusions~5.7 Gb PF potato transcriptome sequence (3 varieties)

~14.3 Gb PF tomato transcriptome sequence (6 varieties)

DM1-3 516R44 draft genome is an excellent scaffold for potato and tomato GAII transcriptome alignments.

SNPs are not evenly distributed in genes/genomes

Genes with signatures of selection (Ka/Ks; high FST) tend to be genes associated with response to abiotic and biotic stress.

Co-adapted complexes result from selection during plant breeding.

Lessons Learned: Control GAII Sequence of DM1-3 516R44 would permit bioinformatic optimization or pipelines rather than relying on empirical validation.

Acknowledgments

Collaborators, OSUMatt Robbins

Sung-Chur SimTroy Aldrich

Collaborators, CAUWencai Yang

Collaborators, CAASSanwen Huang

FundingUSDA/AFRIThis project is supported by the Agriculture and Food Research Initiative of USDA’s National Institute of Food and Agriculture.

Collaborators, MSUDavid DouchesC Robin BuellJohn Hamilton

Kelly Zarka

Collaborators, CornellWalter de JongLucas MuellerJoyce van Eck

Collaborators, UCDAllen Van Deynze

Kevin StoffelAlex Kozic