Comparative analyses of the potato and tomato transcriptomes

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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

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

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) 400 300 Data Collection Assembly Analysis: transcriptome complexity SNP calling/validation identification of genes under selection

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

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

Velvet Assemblies of Potato Illumina Sequences Alignment of S. tuberosum GAII-transcriptome contigs to the PGSC draft genome sequence from DM1-3 516R44: 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)

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

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

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

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

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

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

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

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)

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

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

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

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

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

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.

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.