danforth 2015

Adaptation in maize: domestication and beyond

Jeffrey Ross-Ibarra @jrossibarra • www.rilab.org

Dept. Plant Sciences • Center for Population Biology • Genome Center University of California Davis

photo by lady_lbrty

Brandon Gaut

Hocholdinger & Hoecker 2007 TIP Genetics

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Meyerowitz 1994 Current Biology Duvick et al. 1999 US 6639132 B1

maize origins

Tripsacum extinct maizeF1F1

teosinte (Z. mays ssp. parviglumis)

maize (Z. mays ssp. mays)

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Beadle 1979 Field Museum of Nat. Hist. Bull.

Briggs et al. 2007 Genetics

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Wang et al. 2005 Nature

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Figure 1.Phenotypes. a. Maize ear showing the cob (cb) exposed at top. b. Teosinte ear with the rachisinternode (in) and glume (gl) labeled. c. Teosinte ear from a plant with a maize allele of tga1introgressed into it. d. Close-up of a single teosinte fruitcase. e. Close-up of a fruitcase fromteosinte plant with a maize allele of tga1 introgressed into it. f. Ear of maize inbred W22(Tga1-maize allele) with the cob exposed showing the small white glumes at the base. g. Earof maize inbred W22:tga1 which carries the teosinte allele, showing enlarged (white) glumes.h. Ear of maize inbred W22 carrying the tga1-ems1 allele, showing enlarged glumes. For highermagnification copies of f–h see Supplementary Information.

Wang et al. Page 10

Nature. Author manuscript; available in PMC 2006 May 23.

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teosinte maizeClint Whipple, BYU

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Studer et al. 2011 Nat. Gen.; Vann et al. 2015 PeerJ

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mutation rate21, strongly suggesting that the Hopscotch insertion (and thus, the older Tourist as well) existed as standing genetic variation in the teosinte ancestor of maize. Thus, we conclude that the Hopscotch insertion likely predated domestication by more than 10,000 years and the Tourist insertion by an even greater amount of time.

We identified four fixed differences in the portion of the proximal and distal components of the control region that show evidence of selection. We used transient assays in maize leaf protoplasts to test all four differences for effects on gene expression. Maize and teosinte chromosomal segments for the portions of the proximal and distal components with these four differences were cloned into reporter constructs upstream of the minimal promoter of the cauliflower mosaic virus (mpCaMV), the firefly luciferase ORF and the nopaline synthase (NOS) terminator (Fig. 4). Each construct was assayed for luminescence after transformation by electroporation into maize pro-toplast. The constructs for the distal component contrast the effects of the Tourist insertion plus the single fixed nucleotide substitution that distinguish maize and teosinte. Both the maize and teosinte constructs for the distal component repressed luciferase expression

relative to the minimal promoter alone. The maize construct with Tourist excised gave luciferase expression equivalent to the native maize and teosinte constructs and less expression than the minimal promoter alone. These results indicate that this segment is function-ally important, acting as a repressor of luciferase expression and, by inference, of tb1 expression in vivo. However, we did not observe any difference between the maize and teosinte constructs as anticipated. One possible cause for the lack of differences in expression between the maize and teosinte constructs might be that additional proteins required to cause these differences are not present in maize leaf pro-toplast. Another possibility is that the factor affecting phenotype in the distal component lies in the unselected region between −64.8 and −69.5 kb, which is not included in the construct. Nevertheless, the results do indicate that the distal component has a functional element that acts as a repressor. The functional importance of this segment is supported by its low level of nucleotide diversity (Fig. 3a), suggesting a history of purifying selection.

The constructs for the proximal component of the control region contrast the effects of the Hopscotch insertion plus a single fixed nucleo-tide substitution that distinguish maize and teosinte. The construct with the maize sequence including Hopscotch increased expression of the luciferase reporter twofold relative to the teosinte construct for the proximal control region and the minimal promoter alone (Fig. 4). Luciferase expression was returned to the level of the teosinte con-struct and the minimal promoter construct by deleting the Hopscotch element from the full maize construct. These results indicate that the Hopscotch element enhances luciferase expression and, by

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HKA neutrality tests

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Distalcomponent

Teosinte clusterhaplotype

Maize clusterhaplotype

Proximalcomponent

–65 kbTourist408 bp

Hopscotch4,885 bp

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Figure 3 Sequence diversity in maize and teosinte across the control region. (a) Nucleotide diversity across the tb1 upstream control region. Base-pair positions are relative to AGPv2 position 265,745,977 of the maize reference genome sequence. P values correspond to HKA neutrality tests for regions A–D, as defined by the dotted lines. Green shading signifies evidence of neutrality, and pink shading signifies regions of non-neutral evolution. Nucleotide diversity ( ) for maize (yellow line) and teosinte (green line) were calculated using a 500-bp sliding window with a 25-bp step. The distal and proximal components of the control region with four fixed sequence differences between the most common maize haplotype and teosinte haplotype are shown below. (b) A minimum spanning tree for the control region with 16 diverse maize and 17 diverse teosinte sequences. Size of the circles for each haplotype group (yellow, maize; green, teosinte) is proportional to the number of individuals within that haplotype.

Transient assay constructs

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Figure 4 Constructs and corresponding normalized luciferase expression levels. Transient assays were performed in maize leaf protoplast. Each construct is drawn to scale. The construct backbone consists of the minimal promoter from the cauliflower mosaic virus (mpCaMV, gray box), luciferase ORF (luc, white box) and the nopaline synthase terminator (black box). Portions of the proximal and distal components of the control region (hatched boxes) from maize and teosinte were cloned into restriction sites upstream of the minimal promoter. “ ” denotes the excision of either the Tourist or Hopscotch element from the maize construct. Horizontal green bars show the normalized mean with s.e.m. for each construct.

relative expressionconstruct

gt1

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tb1Figure 2 Map of parviglumis Populations and Hopscotch allele frequency. Map showing the frequencyof the Hopscotch allele in populations of parviglumis where we sampled more than 6 individuals. Size ofcircles reflects number of individuals sampled. The Balsas River is shown, as the Balsas River Basin isbelieved to be the center of domestication of maize.

as our independent trait for phenotyping analyses. SAS code used for analysis is available athttp://dx.doi.org/10.6084/m9.figshare.1166630.

RESULTSGenotyping for the Hopscotch insertionThe genotype at the Hopscotch insertion was confirmed with two PCRs for 837 individualsof the 1,100 screened (Table S1 and Table S2). Among the 247 maize landrace accessionsgenotyped, all but eight were homozygous for the presence of the insertion Withinour parviglumis and mexicana samples we found the Hopscotch insertion segregatingin 37 (n = 86) and four (n = 17) populations, respectively, and at highest frequencywithin populations in the states of Jalisco, Colima, and Michoacan in central-westernMexico (Fig. 2). Using our Hopscotch genotyping, we calculated diVerentiation betweenpopulations (FST) and subspecies (FCT) for populations in which we sampled sixteenor more chromosomes. We found that FCT = 0, and levels of FST among populationswithin each subspecies (0.22) and among all populations (0.23) (Table 1) are similar togenome-wide estimates from previous studies Pyhajarvi, HuVord & Ross-Ibarra, 2013.Although we found large variation in Hopscotch allele frequency among our populations,BayEnv analysis did not indicate a correlation between the Hopscotch insertion andenvironmental variables (all Bayes Factors < 1).

Vann et al. (2015), PeerJ, DOI 10.7717/peerj.900 8/21


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NATURE GENETICS ADVANCE ONLINE PUBLICATION 3

L E T T E R S

mutation rate21, strongly suggesting that the Hopscotch insertion (and thus, the older Tourist as well) existed as standing genetic variation in the teosinte ancestor of maize. Thus, we conclude that the Hopscotch insertion likely predated domestication by more than 10,000 years and the Tourist insertion by an even greater amount of time.

We identified four fixed differences in the portion of the proximal and distal components of the control region that show evidence of selection. We used transient assays in maize leaf protoplasts to test all four differences for effects on gene expression. Maize and teosinte chromosomal segments for the portions of the proximal and distal components with these four differences were cloned into reporter constructs upstream of the minimal promoter of the cauliflower mosaic virus (mpCaMV), the firefly luciferase ORF and the nopaline synthase (NOS) terminator (Fig. 4). Each construct was assayed for luminescence after transformation by electroporation into maize pro-toplast. The constructs for the distal component contrast the effects of the Tourist insertion plus the single fixed nucleotide substitution that distinguish maize and teosinte. Both the maize and teosinte constructs for the distal component repressed luciferase expression

relative to the minimal promoter alone. The maize construct with Tourist excised gave luciferase expression equivalent to the native maize and teosinte constructs and less expression than the minimal promoter alone. These results indicate that this segment is function-ally important, acting as a repressor of luciferase expression and, by inference, of tb1 expression in vivo. However, we did not observe any difference between the maize and teosinte constructs as anticipated. One possible cause for the lack of differences in expression between the maize and teosinte constructs might be that additional proteins required to cause these differences are not present in maize leaf pro-toplast. Another possibility is that the factor affecting phenotype in the distal component lies in the unselected region between −64.8 and −69.5 kb, which is not included in the construct. Nevertheless, the results do indicate that the distal component has a functional element that acts as a repressor. The functional importance of this segment is supported by its low level of nucleotide diversity (Fig. 3a), suggesting a history of purifying selection.

The constructs for the proximal component of the control region contrast the effects of the Hopscotch insertion plus a single fixed nucleo-tide substitution that distinguish maize and teosinte. The construct with the maize sequence including Hopscotch increased expression of the luciferase reporter twofold relative to the teosinte construct for the proximal control region and the minimal promoter alone (Fig. 4). Luciferase expression was returned to the level of the teosinte con-struct and the minimal promoter construct by deleting the Hopscotch element from the full maize construct. These results indicate that the Hopscotch element enhances luciferase expression and, by

a

b

0.06

A B C D M

T

P = 0.95 P = 0.41 P = 0.04

HKA neutrality tests

P 0.0001

0.04

0.02

0–67 kb –66 kb

Distalcomponent

Teosinte clusterhaplotype

Maize clusterhaplotype

Proximalcomponent

–65 kbTourist408 bp

Hopscotch4,885 bp

–64 kb –58 kb

Figure 3 Sequence diversity in maize and teosinte across the control region. (a) Nucleotide diversity across the tb1 upstream control region. Base-pair positions are relative to AGPv2 position 265,745,977 of the maize reference genome sequence. P values correspond to HKA neutrality tests for regions A–D, as defined by the dotted lines. Green shading signifies evidence of neutrality, and pink shading signifies regions of non-neutral evolution. Nucleotide diversity ( ) for maize (yellow line) and teosinte (green line) were calculated using a 500-bp sliding window with a 25-bp step. The distal and proximal components of the control region with four fixed sequence differences between the most common maize haplotype and teosinte haplotype are shown below. (b) A minimum spanning tree for the control region with 16 diverse maize and 17 diverse teosinte sequences. Size of the circles for each haplotype group (yellow, maize; green, teosinte) is proportional to the number of individuals within that haplotype.

Transient assay constructs

mpCaMV luc

luc

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Figure 4 Constructs and corresponding normalized luciferase expression levels. Transient assays were performed in maize leaf protoplast. Each construct is drawn to scale. The construct backbone consists of the minimal promoter from the cauliflower mosaic virus (mpCaMV, gray box), luciferase ORF (luc, white box) and the nopaline synthase terminator (black box). Portions of the proximal and distal components of the control region (hatched boxes) from maize and teosinte were cloned into restriction sites upstream of the minimal promoter. “ ” denotes the excision of either the Tourist or Hopscotch element from the maize construct. Horizontal green bars show the normalized mean with s.e.m. for each construct.

relative expressionconstruct

gt1

hard sweep

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GGTCGA ATG ACT GAT CCA CAT CGA CTG TAG

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GGTCGA ATG ACT GAT CCA CAT CGA CTG TAG

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Vann et al. 2015 PeerJ

polygenic adaptation

30% phenotypic variance

0% phenotypic variance

Hufford et al. 2012 Nat. Gen. Chia et al. 2012 Nat. Gen

13 teosinte 23 maize

~500 genes (2%) 11M shared SNPs

3,000 fixed genomes:

Hufford et al. 2012 Nat. Gen. Chia et al. 2012 Nat. Gen

13 teosinte 23 maizegenomes:

Swanson-Wagner et al. 2012 PNAS

whereas others are lost after domestication (Fig. 3B). It should benoted that many of these genes have unique coexpression edges inmaize that are not observed in teosinte (Fig. S4B).

Expression data provide an opportunity to investigate furtherfunctional alterations to genes located within genomic regionsthat population genomic analyses identify as targets of selective

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GRMZM2G137947

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Fig. 3. Analysis of genes with altered expression or conservation and targets of selection during improvement and/or domestication. (A) Venn diagramshowing the overlap between DE genes, AEC genes, and the genes that occur in genomic regions that have evidence for selective sweeps during maizedomestication or improvement (Dom/Imp genes). (B) Teosinte coexpression networks for three genes (GRMZM2G068436, GRMZM2G137947, andGRMZM2G375302). (Right) Edges that are maintained in maize coexpression networks are shown. Although the differentially expressed gene (red node) ishighly connected in teosinte, most of these connections are lost in maize. However, some parts of the teosinte network are still conserved in maize. (C) Cross-population composite likelihood ratio test (XP-CLR) plot shows the evidence for a selective sweep that occurs on chromosome 9. The tick marks along the xaxis represent genes, and the red tick mark indicates the gene (GRMZM2G448355) that was chosen as the candidate target of selection and is differentiallyexpressed in maize and teosinte. The bar plot underneath the graph shows the expression levels of all maize (blue) and teosinte (red) samples. (D) XP-CLR plotfor a large region on chromosome 5. The candidate target of selection is indicated in green and shows similar expression in maize and teosinte. Two othergenes (red) exhibit DE. (E) Neighbor-joining tree shows the relationships among the haplotypes at GRMZM2G141858. (Right) Bar plot shows expression levelsfor each genotype; red bars indicate teosinte genotypes, and blue bars represent maize genotypes. At least one teosinte genotype (TIL15) contains thehaplotype that has been selected in maize and has expression levels similar to maize genotypes.

Table 2. Genes in selected regions with evidence for DE or AEC

Gene listNo. genes selectedduring dom/imp

% up-regulatedin maize Significance

% higher connectedin maize % candidates

AEC and DE (n = 276) 46 76 0.0002 41.3 39.1DE only (n = 336) 44 61 0.0230 40.9 22.7AEC only (n = 839) 89 54 0.1837 57.3 32.6

dom, domestication; imp, improvement.

4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1201961109 Swanson-Wagner et al.

Beissinger et al. In Prep

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how to adapt: domestication

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regulatory variation

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maize

Mexico lowland

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Matsuoka et al. 2002; Piperno 2006 Perry et al. 2006; Piperno et al. 2009

Mexico highland6,000 BP

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ResultsPatterns of Genetic Structure and Differentiation. Principal com-ponents analysis (PCA) (17) of the maize SNP data identifies 58significant principal components (PCs) (explaining 37.6% oftotal variance), probably reflecting isolation by distance (18) andlinkage effects (19). We use the first nine PCs, which present thestrongest spatial autocorrelation (Fig. S2) and explain a largeportion of the total variance (18.7%), to cluster the accessionsinto 10 geographically distinct groups (Fig. 1A). Meso-Americanmaize falls into three groups: the Meso-American Lowlandgroup, which includes predominantly lowland accessions fromsoutheast Mexico and the Caribbean; the West Mexico group,representing both lowlands and highlands; and the MexicanHighland group, encompassing most of Matsuoka et al.’s high-land Mexican accessions (5) as well as accessions from highlandGuatemala. These clusters also confirm the presence of US-de-rived varieties in South America (20); we excluded these acces-sions from further analysis.In the joint PCA analysis of the three subspecies, the first PC

(10.8% of variance) separates maize from its wild relatives andconfirms the similarity between maize from the Mexican Highlandgroup and parviglumis (Fig. 1B). The second PC (4.8%of variance)mainly separates the genetic groups of maize along a north–southaxis, with the Northern United States and Andean Highlands atthe extremes. The third PC (2.7% of variance) predominatelyreflects the difference between parviglumis and mexicana. TheMexican Highland cluster extends toward mexicana along bothPC 1 and 3, suggesting that the similarity of highland maize toparviglumis may reflect admixture with mexicana.

Admixture Analysis. Simulation of gene flow of mexicana into theMeso-American Lowland maize group suggests that 13% cu-mulative historical introgression is sufficient to explain observeddifferences between lowland and highland maize in terms ofheterozygosity and differentiation from parviglumis (Fig. S3).Structure analysis (21) of all Mexican accessions lends supportfor this magnitude of introgression (Fig. 2). The three subspeciesform clearly separated clusters, but evidence of admixture is

evident in all three groups, and the two wild relatives show clearsigns of bidirectional introgression at altitudes where theirranges overlap (Fig. 2). Highland maize shows strong signs ofmexicana introgression, with 20% admixture observed in theMexican Highland cluster, but below 1,500 m mexicana in-trogression drops to less than 1%. Introgression from parviglumisinto maize is much lower overall, reaching its highest averagevalue (3%) in the lowland West Mexico group.

Drift Analysis. Because introgression from mexicana may affectancestry inference based on genetic distance from parviglumis, wetook an approach that does not require reference to the wild rel-atives. Under models of historical range expansion, genetic dif-ferentiation increases away from the population of origin (22, 23),and estimates of drift from ancestral frequencies have been appliedsuccessfully to identify ancestral populations (24). We thereforeapplied the method of Nicholson et al. (25) to estimate simulta-neously ancestral frequencies and F, a measure of genetic drift ofaway from these frequencies, for sets of predefined populations.To illustrate the potential impact ofmexicana introgression, we

first performed a standard analysis that includes each maizepopulation in turn in conjunction with the two wild relatives.Average drift away from the inferred common ancestor of maize,parviglumis, and mexicana is higher for maize (F = 0.24) than formexicana (F = 0.15) or parviglumis (F = 0.07), probably due tochanges in allele frequency following the domestication bottle-neck. Because the inferred ancestral frequencies are closer tothose of the wild relatives than to present-day maize, comparisonwith this ancestor is sensitive to introgression from these sub-species. It therefore is not surprising that estimates of F betweenindividual maize populations and the common ancestor of allthree taxa identify the Mexican Highland group as being mostsimilar (Fig. 3A). This pattern is maintained in an analysis ex-cluding mexicana, in which Mexican Highland maize is tied withtheWestMexico group as themost ancestral population (Fig. 3B).To mitigate the impact of introgression, we used a slightly

modified approach that excludes both parviglumis and mexicanaand calculates genetic drift with respect to ancestral frequenciesinferred from domesticated maize alone. Because the genetic

Fig. 1. (A) Map of sampled maize accessions colored by genetic group. (B) First three genetic PCs of all sampled accessions.

van Heerwaarden et al. PNAS | January 18, 2011 | vol. 108 | no. 3 | 1089

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domestication in Mexico lowland

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Introgression

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Lauter et al. 2004 Genetics



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scal

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• selection from standing variation when 2Neµ < 1

Messer and Petrov 2013 TIG



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scal

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M T G P H R L

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M T N P H R L

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M T G P H R L


M T N P H R L


x x x

M T G P H R L


M T N P H R L


x xx x



scal

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Ne ~ 2,000,000 Ne ~ 600,000

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scal

ed d

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Ne ~ 150,000 Ne ~ 10,000*

Ne ~ 2,000,000 Ne ~ 600,000µ ∝ 130 x 106bp µ ∝ 220 x 106bp

µ ∝ 2,500 x 106bp µ ∝ 3,100 x 106bp

2Neµ > 1

2Neµ < 1

Brandon Gaut

maizeArabidopsisKew C-Value Database

log 1C genome size

Bilinski et al. In Prep

2.50

2.75

3.00

3.25

3.50

3.75

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Gen

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Size

(Gb)

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knob 350 (Tr1)

knob 180

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knob 180knob 350

32 TE familiesoverall TE contenttotal genome size


genome size


knob 180knob 350

32 TE familiesoverall TE contenttotal genome size


genome size


total genome size

Rayburn et al. 1994 Plant Breeding Francis et al. 2008. Ann. Bot.

cycle time that did not exceed 20 h compared with a muchgreater spread of cycle times for the monocots. If DNAmass per se is the limiting factor for cell cycle time, wehypothesize that cycle times would be the same for dicotsand monocots of comparable C-value. This is so even ifthe data for Scilla sibirica and Trillium grandiflorum are

excluded. Indeed, if we ignore the marked discontinuityof the y-axis caused by their inclusion, then the nucleotypiceffect is strong for all species regardless of phylogeny. Totest the rigour of these hypotheses would require data toplug the gap between Trillium grandiflorum and themajority of C-value/cell cycle times analysed here.

Separate plots for diploids and polyploids show a strongnucleotypic effect on CCT in diploids (Fig. 3; Table 2).Removing the five diploid outliers (.25 pg) reduced theslope (b ¼ 0.27) by approximately four-fold but theregression continued to be significant (P , 0.001). Forthe polyploids, a nucleotypic effect on CCT was alsodetected (Fig. 3; Table 2); however, removing the two poly-ploid outliers rendered the regression non-significant (y ¼0.03x 2 13.5). This confirms previous work in which theslope/rate of increase in CCT with increasing DNA washigher in diploids than in autopolyploids (Evans et al.,1972). With the exception of Scilla sibirica, CCT in poly-ploids is generally more buffered than in diploids (Fig. 3).

We acknowledge that some traditionally classifieddiploids are not necessarily so (see Soltis and Soltis,1999). For example, there are strong arguments that Zeamays is actually an allotetraploid (2n ¼ 4x ¼ 20; Gaut andDoebly, 1997). However, in the data reported here wehave assigned ploidy level as listed by the authors of thepapers and reviews we have consulted.

The longest CCTs (.20 h) are exhibited by the peren-nials (Fig. 4). Indeed, the data for perennials overall had anearly seven-fold steeper slope (b ¼ 1.37) than a compar-able regression for annuals (b ¼ 0.20; Table 2). Thesedata are consistent with findings of Bennett (1972) wherethe mean CCT in 19 annuals was significantly shorterthan in eight obligate perennials. Where our analysesdiffer from Bennett (1972) is in relation to the broadrange of CCTs shown by perennials compared withannuals (Fig. 4). However, in Fig. 4 the longer CCTs

FI G. 3. DNA C-value (pg) and cell cycle time (h) in the root apical mer-istem of a range of diploid and polyploid angiosperms. See Table 2 for

regression analyses.

FI G. 2. DNA C-value (pg) and cell cycle time (h) in the root apical mer-istem of a range of (A) eudicots and monocots (n ¼ 110), and (B) eudicots

(n ¼ 60). See Table 2 for regression analyses.

TABLE 2. Regression analyses of all data presented inFigs. 2–4 together with the percentage variance accountedfor by the regression (R2), the level of probability (P) for

each regression

Regression (y ¼ bx þ a) R2 P n

All measurements y ¼ 1.09x þ 5.39 54.2 *** 110Monocots y ¼ 1.29x þ 2.44 58.7 *** 48Eudicots y ¼ 0.32x þ 10.2 15.4 *** 62Diploids y ¼ 1.04x þ 4.95 49.86 *** 86Polyploids y ¼ 1.14x þ 3.12 56.3 *** 24Annuals y ¼ 0.20x þ 10.7 19.9 *** 75Perennials y ¼ 1.37x þ 4.13 63.6 *** 35

*** P , 0.001; n, number of replicates.

Francis et al. — DNA C-value and the Cell Cycle750

at University of C

alifornia, Davis - Library on February 19, 2013

http://aob.oxfordjournals.org/D

ownloaded from

Francis et al. 2008. Ann. Bot.

0

10

20

30

100 105 110DNA

plants

cycle0

6

late flowering

early flowering

• “Soft sweeps” and polygenic selection predominate in maize

• Gene flow may provide a novel source of adaptive alleles

• Both population size and mutational target contribute

• Large, complex genomes may mean more targets and more soft sweeps in plants

• Genome size itself may be adaptive

Concluding Thoughts

Acknowledgments

Maize Diversity GroupPeter Bradbury

Ed Buckler John Doebley Theresa Fulton

Sherry Flint-Garcia Jim Holland

Sharon Mitchell Qi Sun

Doreen Ware

CollaboratorsJim Birchler Jeremy Berg

Graham Coop Nathan Springer

Lab AlumniTim Beissinger (USDA-ARS, Mizzou)

Matt Hufford (Iowa State) Tanja Pyhäjärvi (Oulu)

Shohei Takuno (Sokendai) Joost van Heerwaarden (Wageningen)