Genetic Diversity and the Effects of Artificial Selection in Maize

1. Genetic Diversity and the Effects of Artificial Selection in Maize

3. Maize Diversity Project Team

4. Molecular Diversity How has selection shaped molecular diversity in maize? What is the relationship of selected genes to agronomic traits? Goal: Identify genes exhibiting selection • Domestication, agronomic improvement, and local adaptation Community resource: SNP marker collection

5. Teosinte Landraces Inbreds/Hybrids Photos courtesy J. Doebley

7. Major predictions for the model Those genes have contributed most to maize improvement, i.e. have experienced the strongest history of selection have the least genetic variability left to contribute to crop improvement by classical breeding. These genes will not be detected in standard QTL experiments because all lines will contain similar alleles.

8. Can we develop genomics screens to identify genes that have undergone selection? Invariant SSR approach (Vigouroux et al. 2002 PNAS 99:9650) Directly contrast sequence diversity among teosintes and inbreds (Wright et al. 2005 Science 308:1310) Are genes with low inbred diversity enriched for selected genes? (Yamasaki et al. 2005 Plant Cell 17:2859) mcmullenm@missouri.edu for .pdfs

9. Summary of Sequencing on Random Genes(Irie Vroh Bi, Masanori Yamasaki, Kate Houchins) MPZ inbreds – (temperate) B73(2), Mo17(2), Hp301, Il14H, Ky21, M37W, Oh43, (tropical) CML69, CML247, CML322, CML333, KUI3, KUI11, NC350. 1095 alignments - 6169 SNPs. MPZ inbreds + 16 teosinte partial inbreds 774 alignments – 3463 SNPs MPZ inbreds – 6136 SNPs in teosintes.

11. Sequence statistics for 1095 genes for diverse maize inbred lines. N L Total L S Total S π All Maize 13.1 280.4 307034 5.6 6169 0.0067 Temperate6.7 292.2 310306 4.3 4560 0.0065 Tropical 6.6 290.8 308816 4.2 4427 0.0061 N = number of sequences, L = length of alignment, S = number of segregating sites, π average number of pairwise differences per bp.

12. Inbred-Teosinte Sequence Summary • Number of alignments >5 in both sets 774 • Average sample size inbreds 12.0 • Average sample size teosinte 12.7 • Average alignment length 294 • Total SNPS in inbreds 3463 • Total SNP in teosintes 6136

13. Diversity in maize inbreds vs. teosinte 0.07 0.06 0.05 0.04 q inbreds 0.03 0.02 0.01 0 0 0.02 0.04 0.06 0.08 q teosintes Average q.inbred/q.teosinte 0.57 Excluding q.inbred=0 values 0.63

14. To identify the selected genes we need new statistical approaches • There are two models: a selection model and a bottleneck model • We must estimate the size of the bottleneck • For each model, we estimate the probability of the model given the data (the likelihood) for each gene • This is very simulation and computer intensive! • This approach allows us to estimate the proportion of genes under selection and to identify the candidates

15. Na t1 Nb Na t2 Np t1 Nb t2 Np Two models: To be considered selected need to fail the neutral model and be accepted by the selected model. selected neutral

16. Genes significant for selection

17. On a genomic scale…. • Assume 40,000 genes in maize • 40,000 x 0.04 = 1600 selected genes • Before genome scans, 11 genes had been identified as selected by population genetic approaches • By sequencing 1000 genes, have ~30 novel candidates • These genes need to be divided between domestication and improvement

18. What genes show evidence of selection? • Genes involved in amino acid synthesis or metabolism • Genes involved in growth response. • Transcription factors and signal transduction components. • Unique genes with no significant BLAST homologies.

19. Are genes with low inbred diversity enriched for domestication and improvement candidates?(Masanori Yamasaki) Chose 35 genes with no diversity among the MPZ inbred set. Sequenced same region in 16 haploid landrace samples, 16 teosinte partial inbreds and a Tripsacum dactyloides sample. Performed Hudson-Kreitman-Aguadé(HKA) (tests for selection) on inbreds, landraces and teosintes against the neutral genes adh1, glb1, fus6 and bz2. Performed coalescent simulations of domestication (CS) of inbreds vs. teosintes and landraces vs. teosintes.

21. ARF Amino Acid Transporter 0.01 0.02 0.01 0 1 1000 2000 3000 0 1 500 1000 Unknown GTP-binding Protein 0.02 0.02 0.01 0.01 0 0 1 500 1000 1500 1 500 1000 1500 Ankyrin repeat F-box (circadian clock) 0.03 0.08 0.06 0.02 0.04 0.01 0.02 0 0 1 1000 2000 3000 1 1000 2000 0.02 Fruit protein Chromatin remodeling 0.03 0.02 0.01 0.01 0 0 1 1000 2000 3000 1 500 1000 1500 p Nucleotide position (bp)

23. Do genes exhibiting signatures of selection control agronomic traits?(Sherry Flint-Garcia) • Hypothesis: manipulation of the expression of domestication and improvement genes will alter key agronomic traits • Methods: use genetic and transgenic approaches to examine teosinte, exotic, and inbred alleles • Test case: amino acid composition in kernels • Evidence for selection for cysteine synthase, chorismate mutase, dihydrodipicolinate synthase and hexokinase

24. To what extend has diversity in amino acid synthesis genes been reduced by selection? (Sherry Flint-Garcia) • Whitt et al., 2002 demonstrated that 3 of 6 genes in starch synthesis pathway in maize show solid evidence of artificial selection • Evidence for selection for cysteine synthase, chorismate mutase, dihydrodipicolinate synthase and hexokinase from random sequencing • Chose 16 additional genes for important steps in amino acid synthesis, sequenced in teosintes, landraces and inbreds and conducted tests of selection

25. 30 25 Teosinte (n = 7) 25 Landraces (n = 11) 20 Maize (n = 27) 20 15 15 Percent of Kernel Weight Percent of total amino acid 10 10 5 5 0 0 Teosinte vs. Landraces ** ** ** ** ** ns ns ** ns ** ** ** ** ** ** ns ** ** ** Teosinte vs. Inbred Lines ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ns ** ns ** Valine Lysine Serine Proline Alanine Glycine Leucine Arginine Histidine Tyrosine Cysteine Total Amino Acid Isoleucine Threonine Methionine Tryptophan Aspartic Acid Glutamic Acid Phenylalanine

26. Trans-cinnamic acid Lignin PAL Glucose Phenylalanine Tyrosine Glycine Serine O-Acetylserine 3-Phospho- glycerate Prephenate Cysteine synthase Erythrose 4-P Chorismate mutase Cysteine 2-isopropyl- malate synthase Phosphoenol pyruvate DAHP Shikimate Chorismate Leucine Anthranilate Synthase β Pyruvate Alanine Pyruvate Anthranilate Valine Acetyl-CoA Acetohydroxy acid synthase Indole-3-glycerol phosphate Asparagine Isoleucine Tryptophan Synthase β1 Aspartate Amino- transferase Asparagine synthetase Tryptophan 2-Ketobutyrate Aspartate Oxalo- acetate Threonine deaminase TCA Cycle Aspartate kinase Glutamate Threonine Aspartate 4-seminaldehyde NH4 Arginine α-Keto- glutarate DHDP synthase Proline Homoserine 4-phosphate Proline dehydrogenase Glutamate dehydrogenase Cysteine 2,3-Dihydro- dipicolinate Cystathionine γ-synthase Glutamate Cystathionine Homocysteine Lysine Glutamine NH4 NO3– NO2– Methionine Nitrate Reductase Histidine SAM synthetase I SAM synthetase II Hexokinase (N:C sensing) S-Adenosyl- methionine ntl1 --nitrogen regulating protein

27. Sequencing candidate genes • Goal is to sequence 1000 candidate genes in all inbreds for the 25DL, 16 teosintes, 2 Tripsacum, and W22 R-std • Shared responsibility by E. Buckler and M. McMullen laboratories • Develop SNP (or sequence) based assays for association analysis • Develop a mechanism to accept candidate gene suggestions for outside the project • www.panzea.org

28. 100% 80% 60% 38,000 genes 1,000 genes 1,000 genes

29. Implications for GEM • For the vast majority of genes inbreds lines retain on average 60% of common diversity of teosinte and 80% of the diversity of landraces. Therefore the problem of loss of diversity is a specific problem to particular genes and traits rather than a general problem • Most of the diversity lost in unselected genes is in rare alleles and therefore hard to capture

30. Implications for GEM • Our studies to date have not addressed specific adaptation, possibly a more important justification for GEM than limited diversity per se • It is hard for me to think about how to tap diversity for specific adaptation without considering diversity in a trait context.