1. Comparative analyses of the potato and tomato transcriptomes
David Francis, AllenVan Deynze, John Hamilton, Walter De Jong, David Douches, Sanwen Huang, and C. Robin Buell
Supported by the AFRI Plant Breeding, Genetics, and Genomics Program of USDA’s National Institute of Food and Agriculture

2. 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 DM R44 (S. tuberosum L. Phureja group); S. tuberosum, S. lycopersicum, S. pimpinellifolium GAII transcriptomes
Technology
Next Generation Sequencing
SNP genotyping

3. What comparisons do we want to make?
How well do S. tuberosum expressed sequences align to DM R44 genomic sequences?
How well do S. lycopersicum expressed sequences align to DM R44 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?

4. Library creation/QC GAII sequencing (single and paired end)
400
300
Data Collection Assembly
Analysis:
transcriptome complexity
SNP calling/validation
identification of genes under selection

5. Illumina GA II Output for Potato
Sample
Total Clusters
Total 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

6. Velvet Assemblies of Potato Illumina Sequences
With a minimum kmer of 31 and a minimum contig length of 150bp:
Variety
Total Gb
Transcriptome 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
58754
358
6.9

7. Velvet Assemblies of Potato Illumina Sequences
Alignment of S. tuberosum GAII-transcriptome contigs to the PGSC draft genome sequence from DM R44:
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)

8. Tomato Illumina GA II Output
Variety
Insert Size
Read Length
Total Reads
PF Reads
%PF Passed
Total PF
FL7600
300
61/47
22,491,304
20,685,342
92.0 60
16,025,976
14,382,577
89.8
15,645,164
13,985,875
89.4
49,053,794
NC84173
350
61/61
27,079,946
22,687,626
83.8
11,058,431
10,366,811
93.8
14,401,240
12,687,134
88.1
52,539,617
OH9242
26,960,898
24,874,218
92.3
10,316,775
9,671,753
14,676,814
12,879,812
87.8
51,954,487 T5
26,799,944
24,677,302
92.1
16,822,639
14,738,351
87.6
15,726,257
13,744,511
87.4
59,348,840
PI114490
17,721,226
16,422,842
92.7
17,115,349
14,902,672
87.1
17,890,649
15,248,587
85.2
52,727,224
PI212816
17,631,906
16,450,422
93.3
18,238,179
15,354,882
84.2 84
21,829,622
18,500,235
84.8
57,699,707

9. Velvet Assemblies of Tomato Illumina Sequences
With a k-mer length of 31 and a minimum contig length of 150bp:
Variety
Total Gb
Transcriptome 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 41
61,310
690
11.7
PI212816
3.00
41.1
66,118
471

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

11. Alignment of contigs relative to DM1-3 516R44
FL7600 (93.7 % id; 94.4 % coverage)
Snowden (97.9; 94.7)

12. Identify intra-varietal SNPs
Query
SNPs
Filtered SNPs
Atlantic Asm
224748
150669
Premier Asm
265673
181800
Snowden Asm
258872
166253
A/C SNP

13. Filtered SNP counts Filtering on SNP quality and 1 SNP/ 150bp window
Ref
Query
d 10
d 20
d 30
d 40
d 50
d 60
d 100
atlantic
21336
17509
14493
12150
10277
8673
4435
premier
21789
18050
15084
12477
10584
8919
4620
snowden
19997
16518
13694
11378
9689
8048
4173
21117
17096
14106
11785
9790
8222
4228
22951
18431
15016
12377
10300
8703
4371
20972
16846
13709
11357
9479
7873
4113
20777
16998
13984
11619
9647
8131
4186
22101
17888
14701
12068
10124
8650
4223
21083
16963
13792
11218
9359
7735
3896

14. Filtered SNP counts No. SNPs Validation rate depth of coverage
Filtering on SNP quality and 1 SNP/ 150bp window
No. SNPs
Validation
rate
depth of coverage

15. Genotyping platforms….
Comments on quality control…
Data….
direct comparison of sequence
analysis of SNPs across populations

16. COS
R-gene
Comparison of two genes on tomato chromosome 9 BAC

17. 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%)

18. 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%)

19. 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)

21. Population structure: coding vs. non-coding
Processing
Fresh-market
Vintage
Landrace
All 173 markers (K=6)
CA & OH OH CN
89 Coding markers (K=5)
84 Non-coding markers (K=6) CA OH OH CN
500K burnin/750K MCMC reps, 20 runs for each K from 3 to 8 21

22. Distribution of FST for genes
ovate: 0
fw2.2: 0
sp6: 0.14
ovate: 0.26
fw2.2: 0
sp6: 0.73
ovate: 0
fw2.2: 0.5
sp6: 1
ovate: 0
fw2.2: 0.42
sp6: 0.74
ovate: 0.14
fw2.2: 0.46
sp6: 0.05
ovate: 0.31
fw2.2: 0
sp6: 0.47 22

23. Examples of highly polymorphic genes within S. lycopersicum
Note: I am working on a replacement that compares Ka/Ks for selected tomato and potato genes

24. Examples of highly polymorphic genes within S. lycopersicum
Note: I am working on a replacement that compares Ka/Ks for selected tomato and potato genes

25. Distribution of PM genes across populations is not random
Processing Fresh Market Vintage Wild 25

26. Visit us at http://solcap.msu.edu/
Tools, Downloads

27. Conclusions ~5.7 Gb PF potato transcriptome sequence (3 varieties)
~14.3 Gb PF tomato transcriptome sequence (6 varieties)
DM R44 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 DM R44 would permit bioinformatic optimization or pipelines rather than relying on empirical validation.

28. Collaborators, Cornell
Acknowledgments
Collaborators, CAU
Wencai Yang
Collaborators, CAAS
Sanwen Huang
Collaborators, OSU
Matt Robbins
Sung-Chur Sim
Troy Aldrich
Collaborators, Cornell
Walter de Jong
Lucas Mueller
Joyce van Eck
Collaborators, UCD
Allen Van Deynze
Kevin Stoffel
Alex Kozic
Collaborators, MSU
David Douches
C Robin Buell
John Hamilton
Kelly Zarka
Funding
USDA/AFRI
This project is supported by the Agriculture and Food Research Initiative of USDA’s National Institute of Food and Agriculture.