A comprehensive study of the genomic differentiation between temperate Dent and Flint maize
Characterization of the Dent and Flint panels
We genotyped a diverse panel of 136 temperate inbred lines (Additional file 1: Table S1) at high density with the Axiom® Maize Genotyping Array 21]. The array comprises more than 600 k SNP markers, which were identified based on
mid- to high-coverage whole-genome sequence data of 30 representative temperate Dent
and Flint maize lines 21]. Markers were filtered according to quality scores and stable performance on the
array, thus representing high-confidence sequence variants, and their final distribution
followed the average recombination rate along the chromosomes 21]. After stringent quality filtering of the 616,201 markers included on the array,
547,412 high-quality SNPs (88.8 %) remained for analysis. These SNPs tagged 19,759
genes (49.8 % of the annotated gene set of maize) with, on average, two SNPs in their
coding region (52.6 % synonymous and 47.4 % non-synonymous). Slightly more SNPs were
polymorphic in the Flint compared to the Dent panel (95.4 % versus 93.1 %), but the
majority of SNPs segregated in both germplasm pools (88.6 %).
The panel of 136 temperate Dent and Flint inbred lines comprised frequently used and
important founder lines exploited in breeding programs in Europe and the U.S., including
lines which were used as parents for the U.S. and European NAM panels 6], 38], 39]. The 70 Dent lines were selected according to available pedigree information and
their frequency of use and citation 40], 41] to assemble a representative set of lines. Besides 16 European Dent lines, the lines
represent U.S. Corn Belt Dent and include lines from the Maize Association Population
42] and the list of inbred lines with expired U.S. plant variety protection 43]. The 66 Flint lines investigated in this study comprised important founder lines
of European breeding programs like F2 and F7 originating from the French landrace
Lacaune, EP1 from the Spanish landrace Lizargarate, and derivatives of the German
landrace Gelber Badischer Landmais 44]. The Flints comprised in total 34 lines from France, 20 from Germany, four from Spain,
three from Italy, three from North America, as well as one from Switzerland and Austria.
Between the elite lines of the two germplasm pools, we observed a clear separation
of pools (Fig. 1b) and a high genome-wide level of differentiation (F ST
?=?0.14), which is consistent with the long-term genetic differentiation between Dent-type
and Flint-type maize 2], 3].
Genome-wide screens for selection signals
Taking advantage of the characteristics of selective sweeps, we screened the genome
for extreme allele frequencies over extended linked sites to detect regions under
differential selective pressure between Dent and Flint. Signatures of selection in
only one of the two pools, Dent or Flint, were detected based on low levels of nucleotide
diversity (?) 9] and Tajima’s D (TD) 10] in the respective pool. In addition, a signature had to be supported by a high value
of the composite likelihood ratio (CLR) test 11] within the respective pool, which indicates a deviation of the allelic composition
of a genetic region compared to a neutrally evolving sequence determined by the genomic
background. To ensure that the selection signature was specific for one of the two
pools, it had to be associated with a high level of differentiation between Dent and
Flint measured by the fixation index F ST45]. Except for the CLR statistic, which was calculated for non-overlapping grids of
150 kb, we applied a sliding window approach averaging data over windows of 40 SNPs
(sliding by 10 %) and filtered for regions below the 10 % quantile for ? and TD and above the 90 % quantile for F ST
and CLR (Additional file 1: Table S2). Following the approach reported by 17], adjacent windows passing the threshold for all four statistics were grouped together
for candidate gene analysis, as the observed changes in allele frequency were likely
caused by the same selective sweep event. This resulted in a filtered set of 265 windows
for Dent and 158 windows for Flint, with an average length of 331.40 kb and 267.80 kb,
respectively, and thus comparable to the length of domestication windows found in
a previous study 17]. An example of a signature of differential selection in Dent and Flint determined
by all four metrics (?, TD, CLR, and F ST
) is shown in Fig. 1c for a region on chromosome 8 harboring two candidate genes. The underlying genetic
region was composed of four major haplotypes. The first three haplotypes occurred
at intermediate frequencies in Dent, whereas the fourth haplotype was almost exclusive
for Flint.
Genome-wide patterns of diversity and the resulting distribution of selection signatures
in the Dent and Flint panels are given in Additional file 2: Figure S1. Within the filtered set of windows, which covered 4.3 % of the total
length of the maize genome for Dent and 2.1 % for Flint, we identified 876 genes as
candidates under differential selective pressure in Dent and 545 genes for Flint with
14 genes common to both candidate genes sets (Additional file 3: Table S3). This corresponded to 2.2 % and 1.4 % of the filtered gene set of maize,
respectively, and is in the same order of magnitude as the estimated number of genes
under selective pressure during maize domestication and improvement 17]. When comparing the candidate gene sets with the 571 improvement candidates reported
by 17], 26 genes overlapped with the list of Dent candidates but only one gene with the
Flint candidate gene set. Considering that the genetic material studied in 17] comprised mainly U.S. Dent and (sub-) tropical lines and that pool-specific sequence
variation in temperate Dent and Flint has been reported here and, for example, by
5], these results emphasize the relevance of a representative panel of lines belonging
to divergent germplasm pools to obtain a comprehensive picture of the genomic diversity
in maize.
In genome-wide screens for signatures of positive selection, also other forces than
selection, such as heterogeneous mutation and recombination rates along the genome,
past demographic history and background selection shape the genomic diversity and
can give rise to false-positive signals. It is beyond the scope of this paper to infer
a full demographic history of maize for the elite lines and landraces as the breeding
history of maize is complex and violates several assumptions of the classic population
genetics models (e.g. discussed in 46]), as, for example, the assumption of panmictic populations and applicability of the
coalescent at short time scales. We therefore applied the CLR test 11], which detects selective sweeps based on the comparison of the site-frequency spectrum
within a specific genomic region to the average site-frequency spectrum over the genome,
a method which has been successfully used in human and other species to detect selective
sweeps 11], 47], 48]. To further decrease the rate of false-positives, the CLR test was combined with
three additional metrics (?, TD, and F ST
) and we identified signatures of positive selection based on this conservative approach
with an overlap of genome-wide extreme values per metric. The high level of linkage
disequilibrium in temperate Dent and Flint elite lines 21] facilitates the detection of selective sweep signals over sufficiently large genomic
regions by the CLR test. On the other hand, the extent of linkage disequilibrium may
decrease the power to discriminate between signals caused by genetic hitchhiking due
to positive selection and negative background selection in regions with reduced levels
of recombination 49], 50]. To assess the number of false-positives due to this effect, we explored the recombination
landscape in the Dent and Flint panels by estimating lower bounds of historical recombination
events 51]. The proportion of candidate genes located in regions with strongly reduced recombination
rates and high linkage disequilibrium like (peri-) centromeric regions was then estimated.
We found that 74.8 % of the Dent and 80.9 % of the Flint candidates were not located
in regions with low levels of recombination (10 % quantile per chromosome; Additional
file 1: Figure S2) indicating that the majority of candidates represent targets of selection
rather than false-positive signals. Furthermore, in a classic selective sweep scenario
(in contrast to background selection) targets of selection are to be enriched for
derived alleles. As an additional test of our candidate regions, we included information
from Sorghum bicolor to distinguish between ancestral and derived alleles. The Dent and Flint candidate
gene sets revealed significantly higher derived allele frequencies compared to the
remaining genes as measured by Fay and Wu’s normalized H52] (p??2.2e-16; Additional file 1: Table S4), which also supported positive selection as the driving force of the observed
allele frequency changes.
Gene ontology and pathway analyses of candidate gene sets
Considering genetic differentiation and distinct phenotypic characteristics of Dent
and Flint, we tested whether the candidate gene sets were enriched for specific biological
processes or pathways. Gene ontology (GO) terms associated with the identified genes
were available for around 40 % of the candidates (333 for Dent and 214 for Flint).
No significant GO term enrichment of biological processes, cellular components, and
molecular functions could be detected for either of the two sets (Additional file
1: Figure S3). To investigate if candidate genes revealed a pool-specific enrichment
for metabolic pathways, we performed pathway analyses using MapMan 53]. Based on information available for 58 Dent and 40 Flint candidate genes, we observed
a grouping of genes associated with tetrapyrroles (chlorophyll and heme precursors)
for Dent and for terpenoid metabolism for Flint (Additional file 1: Figure S4). The latter included the two genes ZmPPS7.3 (GRMZM2G014508) and ZmPPS8.2 (GRMZM2G483889), which encode a large and a small subunit of the geranyl diphosphate synthase complex
in maize, respectively 54]. Like their homologues in A. thaliana55], they are assumed to be involved in the biosynthesis of precursors of hormones from
the isoprenoid pathway (e.g. gibberellins, brassinosteroids, and abscisic acid). The
ability to produce other downstream products of this enzyme, namely ?-caryophyllenes, has been shown to differ between European Flint and U.S. Dent lines
and suggested that this defense response signal against herbivores was largely lost
in temperate U.S. Dent 56], 57]. The analysis of candidates associated with other traits that are known to differentiate
Dent and Flint revealed six Flint candidates that, according to GO terms, are related
to cold tolerance, a trait that is characteristic for temperate Flint 24]. For two of the candidates, differential expression upon exposure to chilling temperature
has been reported in maize (GRMZM2G03558458] and GRMZM2G09556259]) as well as for the homologous gene of GRMZM2G139680 in rice 60]. The molecular and functional characterization of the identified candidate genes
in maize and the investigation of differences between Dent and Flint in the regulation
of phytohormone pathways or secondary metabolism may provide further insights in the
adaptation of maize to different environments. Up to now, comprehensive RNA expression
data across various developmental stages and tissues are mainly available for U.S.
Dent lines like B73, which underlines the need for a better structural and functional
genomic characterization of the Flint germplasm pool and its unique properties.
Assessing the phenotypic effects of candidate genes on flowering time in a Dent–Flint
introgression library
In the genome-wide selection screens, we identified 18 candidates for Dent and 12
candidates for Flint, which could be assigned to the flowering pathway based on previous
reports in maize, GO terms, and/or sequence homology to flowering genes characterized
in other species 30], 32]–34], 61], 62]. We focused exemplarily on candidate genes associated with the flowering network
in maize as flowering time is an important agronomic trait that differentiates temperate
Dent and Flint. However, functional studies of these genes in maize were available
for only 30 % of the candidates (Additional file 4: Table S5). Here, we investigated the effect of the flowering time candidate genes
in more detail using a maize introgression library.
The introgression library had a Dent genetic background with introgressions from a
Flint donor line and comprised 97 lines, which carried single Flint segments and covered
in total 50.9 % of the Flint donor genome (1048.7 Mb) with a median length of the
donor genome segment size of 10.6 Mb (average: 30.8 Mb; Additional file 5: Table S6). We obtained phenotypic data for male and female flowering time based
on a field experiment carried out at two locations in Germany. Heritabilities were
0.60 (CI
0.95
?=?[0.40; 0.73]) and 0.51 (CI
0.95
?=?[0.27; 0.67]) for male and female flowering time, respectively. Phenotypic differences
between the Dent and Flint parent were larger for male than for female flowering time
(23.2 and 17.8 days, respectively). Based on the least significant difference (??=?0.05),
63 (64.9 %; Fig. 2a) and 16 lines (16.5 %; Fig. 2b) differed significantly from the recurrent Dent parent for male and female flowering
time, respectively. Fifteen of these lines had significant effects for both male and
female flowering time (??=?0.05). When correcting for multiple testing (??=?0.05/97),
six lines (6.2 %) differed significantly for male and none for female flowering time.
Fig. 2. Effect of candidate genes on flowering time in a Dent–Flint introgression library.
Adjusted means of (a) male and (b) female flowering times for 97 introgression lines (circles) and the Dent and the Flint parental line (red and blue squares, respectively). Lines carrying a segment with Dent or Flint flowering time candidate
genes are highlighted in red or blue, respectively, and lines with a Dent and a Flint candidate are shown in black. The dotted and dashed lines represent the significance thresholds without (??=?0.05) and with correction for
multiple testing (??=?0.05/97). Boxplots of adjusted means of flowering times are depicted in the lower parts of (a) and (b) for seven lines carrying Flint haplotypes of Flint flowering time candidates (blue), nine lines carrying Flint haplotypes of Dent flowering time candidates (red), and the 75 lines not carrying a flowering time candidate (gray). For details about the respective lines see Additional file 5: Table S6. Boxplots show the upper and lower quartile, median (horizontal bar), and whiskers (vertical bars) of the adjusted means. Points above and below the whiskers indicate values?±?1.5
times the interquartile range. Significance of Student’s t-tests with p??0.05 is indicated by *
Of the 97 lines, 22 carried a Flint introgression harboring one or several of the
flowering time candidates identified in the selection screens (Additional file 5: Table S6). Fourteen of the 30 candidates were represented in these 22 introgression
lines. Seven lines carried a segment with one or more of seven flowering time candidates
identified in Flint and nine lines carried one or more of six flowering time candidates
identified in Dent. Six lines carried a segment with a combination of Dent and Flint
candidates. Although 75 lines did not carry one of the flowering time candidates identified
in our selection screens, they may carry other flowering time genes with alleles differing
between the Dent and the Flint parent of the introgression library. Lines carrying
the Flint haplotype of a Flint candidate differed significantly from the 75 lines
which did not carry one of the flowering time candidates from the selection screens
(93.1 versus 96.1 days, p value?=?0.011; Fig. 2a). For the lines which carried the Flint haplotype of a Dent selection candidate,
this difference was not significant. The results indicate that in the genetic material
under study, the Flint haplotypes of Flint candidates promoted flowering time more
than the Flint haplotypes of Dent candidates.
Of the six lines with significant difference in male flowering compared to the Dent
parent after correcting for multiple testing (??=?0.05/97), two carried Flint haplotypes
of Flint candidates and one a Flint haplotype of a Dent candidate. One of the lines
included the well-characterized large-effect region comprising the ethylene-responsive
transcription factor Rap2 (related to APETALA2 7, ZmRap2.7, Rap2, GRMZM2G700665) and its regulatory upstream locus Vgt162], a major QTL for flowering time in maize 29], 30]. The other line contained Zcn1 (one of several members of the ZEA CENTRORADIALIS or TERMINAL FLOWER1 (TFL1)-like gene family 32]; also Phosphatidylethanolamine-binding protein1, Pebp1, GRMZM2G092008), for which so far only a moderate effect on flowering time was reported in maize
63]. This gene is related to TFL1 in A. thaliana32], which is an antagonist of the FLOWERING LOCUS T (FT) 64], 65] and required for the maintenance of an indeterminate inflorescence meristem identity
and the regulation of flowering time in A. thaliana and maize 63]–66]. The line with the Dent candidate carried Zmm22 (MADS-transcription factor 69, Mads69, GRMZM2G171650), which was recently reported to be associated with variation in flowering time in
maize 67] and is considered a candidate for maize domestication and/or improvement 68], 69].
Overall, our findings in the introgression library support the relevance of the investigated
genomic regions and their associated candidates for promoting flowering time and confirm
the quantitative nature of flowering time in maize, determined by many genes with
small effects 26] and only few genes with larger effects. Here, the effects of Zcn1 and Zmm22 were stronger than reported previously, which may be attributed to a stronger substitution
effect when replacing a Dent haplotype with a Flint haplotype. We will target potential
expression differences of flowering time candidates in the Dent–Flint introgression
library in future studies to characterize possible differences in the regulation of
the flowering network between germplasm pools adapted to different environments.
Differential selection on components of the flowering network within temperate maize
We investigated the 30 flowering time candidates with respect to their assignment
to endogenous pathways and pathways regulated by environmental factors within the
flowering network to determine if different components of the flowering network were
under selective pressure in Dent and Flint, respectively. Within the flowering network,
Flint candidates were involved predominantly in endogenous signaling, hormone-dependent,
and developmental processes (10 of 12 candidates, 83.3 %), whereas the Dent candidates
indicated a prevalence for response to environmental factors like light and photoperiod
(12 of 18 candidates, 66.7 %; Fig. 3a, Additional file 4: Table S5). As described above, Flint candidates included the well-characterized
Rap2/Vgt1 locus and Zcn1. Furthermore, we found the Squamosa promoter binding protein-transcription factor 25 (Sbp25, GRMZM2G414805) and Gnarley1 (Gn1; also Homeobox protein KNOTTED1-like 4, Knox4, GRMZM2G452178) that are associated with aging and hormone-dependent pathways (Additional file 4: Table S5). Gn1 is likely to act upstream of the “green revolution” gene encoding gibberellin 20-oxidase
70] and to regulate Gibberellin 2-oxidase 1 expression in maize, thus influencing vegetative to reproductive phase transition,
pollen tube growth, and stem elongation by changing the availability of gibberellin
71]. Gibberellin 2-oxidase 1 is additionally regulated by Knotted1 (Kn1, GRMZM2G017087) which was identified as a Dent candidate gene 71]. Another well-characterized Dent candidate is Constans1 (Conz1, GRMZM2G405368), which is a putative ortholog of the photoperiod genes CONSTANS from A. thaliana and Heading date1 in rice 72]. To the best of our knowledge, 20 of the 30 detected flowering time candidates have
not yet been functionally characterized in the context of maize flowering time, but
were associated with the flowering network based on GO terms or reports in other species
such as A. thaliana and rice (Additional file 4: Table S5). Thus, our study revealed candidates that warrant further investigation
of their functional relevance in maize flowering time. Based on the observed allele
frequency differences of the candidate genes within the 136 elite lines and with respect
to their function in maize or, for example, A. thaliana, we hypothesize that different components of the flowering network were under selective
pressure in Dent and Flint. The Flint-specific haplotypes of these genes might constitute
a promising source for the adaptation of maize germplasm pools to shorter vegetation
periods.
Fig. 3. Selection candidates of the maize flowering network and their nucleotide diversity
in 136 elite lines and 38 European landraces. a Candidates associated with the maize flowering network identified under selective
pressure in 70 Dent (red) and 66 Flint (blue) lines based on genotyping data. Candidates are grouped according to their putative
function in endogenous pathways and pathways regulated by environmental factors. For
details about the candidate genes and their classification, see Additional file 4: Table S5. Ambiguous assignments according to GO annotations and literature are indicated
by *. b Nucleotide diversity ? of nine Flint (blue) and 13 Dent (red) flowering time candidate genes for 136 temperate elite lines as well as 31 Flint-type
and seven Dent-type European landraces. Mean values for each gene were calculated
for the panels of Dent and Flint elite lines (left) and for each of the 38 landraces (right). For details about candidate genes, gene-wise ? values, and order of landraces, see Additional file 6: Table S8
Diversity of flowering time candidates in elite lines and European landraces
As most of the European Flint inbred lines are assumed to be derived from few landraces
44], we compared the diversity and the allelic composition of 22 flowering time candidates
(13 Dent and 9 Flint candidates tagged by at least five SNPs) between the elite lines
and a unique panel of 38 European landraces (Additional file 1: Table S7). For each landrace, 22 to 24 plants were genotyped at high density with
the Axiom® Maize Genotyping Array 21]. The majority of the landraces (N?=?31) had Flint-type kernels. These landraces exhibited
lower levels of diversity in the Flint flowering time candidates (gene-wise average:
??=?0.130) compared to the Dent flowering time candidates (??=?0.243), thus confirming the pattern found in the Flint elite lines (Fig. 3b, Additional file 6: Table S8). We further investigated the level of differentiation between Flint-type
landraces and Flint elite lines and observed low levels of F ST
for the Flint flowering time candidates (F ST
?=?0.060; Fig. 4a, FT) with ten landraces from France, Germany, and Spain displaying values even smaller
than 0.050 (Additional file 7: Table S9). These low values of differentiation suggested a major contribution of
the Flint-type landraces to the flowering time candidate gene diversity observed in
the Flint elite lines. This hypothesis was corroborated by the finding that the entire
set of Flint candidate genes also revealed significantly lower levels of differentiation
compared to all other genes, which were not under differential selection between Dent
and Flint elite lines (F ST
?=?0.072 versus 0.095, p value?=?6.0e-04; Fig. 4a, A versus C).
Fig. 4. Differentiation between elite lines (EL) and landraces (LR) for candidate genes. The
upper panel shows the differentiation (F ST
) between 66 Flint elite lines and 31 Flint-type landraces for (a) Flint (blue) and (b) Dent (red) candidate gene sets. The lower panel depicts the differentiation between 70 Dent
elite lines and seven Dent-type landraces for (c) Dent (red) and (d) Flint (blue) candidate gene sets. The boxplots show F ST
values for all (A; gray) genes except the candidates, the candidate (C) genes, and for the subset of candidates
associated with flowering time (FT). Boxplots show the upper and lower quartile, median (horizontal bar), and whiskers (vertical bars) of the F ST
values. Points above and below the whiskers indicate values?±?1.5 times the interquartile
range. Significance of two-sided Wilcoxon rank sum tests with p??0.05 are indicated by * and with p??0.001 by **. For details see Additional file 7: Table S9
Consistent with the hypothesis that Flint elite lines and Flint-type landraces have
a common history, significantly higher levels of differentiation were observed for
Dent candidate genes compared to all remaining genes (F ST
?=?0.111 versus 0.095, p value?=?0.017; Fig. 4b, A versus C). Together, these findings indicated that the reduced diversity observed
for Flint candidate genes in Flint elite lines was already present in a broad panel
of European landraces and that the candidate gene diversity of the Flint elite lines
originate from a limited number of Flint-type landraces used for elite line development
in some historically important breeding centers 44].
The remaining seven landraces displayed at least partially Dent-type kernels. These
landraces revealed high levels of diversity for Dent and Flint flowering time candidates
(??=?0.225 and 0.260, respectively; Fig. 3b, Additional file 6: Table S8) and showed a high level of differentiation with Dent elite lines for the
Dent flowering time candidates (F ST
?=?0.170; Fig. 4c, FT). The same pattern was found in the analysis of the entire Dent candidate gene
set, which revealed significantly higher levels of differentiation compared to all
remaining genes (F ST
?=?0.164 versus 0.111, p value?=?0.026; Fig. 4c, A versus C), but no significant difference for Flint candidates compared to all
remaining genes (F ST
?=?0.138 versus 0.112, p value?=?0.209; Fig. 4d, A versus C; Additional file 7: Table S9). These results indicated that the European Dent-type landraces exhibit
a different allelic composition in the Dent candidates compared to the Dent elite
lines and did most likely not contribute to the Dent elite material under study.
Selection on upstream and genic regions of the candidates
To examine how specific elements of the genic regions contributed to the differentiation
between Dent and Flint, we compared levels of differentiation for 5 kb and 500 bp
upstream regions, genic regions, and exons between the candidate gene sets and all
remaining genes. To increase the resolution of our analyses, we investigated the candidate
gene sets based on whole-genome sequence data of 40 temperate elite lines (21 Dent
and 19 Flint) 21], 22], which were part of the panel of 136 elite lines genotyped with the 600 k array with
the exception of three lines (Additional file 1: Table S1). Based on 13,246,294 bi-allelic SNPs, we observed a significant reduction
of mean ? and TD in 727 Dent and 403 Flint candidate genes tagged by at least five SNPs (of
in total 876 and 545 candidates, respectively) compared to 31,163 remaining genes
(p value??2.2e-16; Additional file 1: Figure S5 and Additional file 1: Table S4). F ST
values calculated between Dent and Flint were significantly higher for candidate gene
sets compared to all remaining genes for 5 kb and 500 bp upstream as well as genic
and exonic regions. Together, these findings supported the results obtained from the
selection screens in the panel of 136 temperate inbred lines genotyped with the 600 k
array.
Previous studies in maize suggested an important role of the divergence of regulatory
elements in the context of domestication 73]–75]. In our study, distributions of F ST
values were comparable for 5 kb and 500 bp upstream as well as genic and exonic regions
in each of the two candidate gene sets (Additional file 1: Figure S6 and Additional file 1: Table S4). However, the power to resolve whether selection acted differentially
in upstream and genic regions was probably limited by the high level of linkage disequilibrium
observed in temperate Dent and Flint lines 21]. The outcome of ongoing large-scale whole genome and transcriptome sequencing will
allow the investigation of the impact of selection on the regulation of gene activity
in the two pools and its consequence for the genomic differentiation between Dent
and Flint.