Comparative SNP and haplotype analysis reveals a higher genetic diversity and rapider LD decay in tropical than temperate germplasm in maize

Understanding of genetic diversity and linkage disequilibrium (LD) decay in diverse maize germplasm is fundamentally important for maize improvement. A total of 287 tropical and 160 temperate inbred lines were genotyped with 1943 single nucleotide polymorphism (SNP) markers of high quality and compared for genetic diversity and LD decay using the SNPs and their haplotypes developed from genic and intergenic regions. Intronic SNPs revealed a substantial higher variation than exonic SNPs. The big window size haplotypes (3-SNP slide-window covering 2160 kb on average) revealed much higher genetic diversity than the 10 kb-window and gene-window haplotypes. The polymorphic information content values revealed by the haplotypes (0.436-0.566) were generally much higher than individual SNPs (0.247-0.259). Cluster analysis classified the 447 maize lines into two major groups, corresponding to temperate and tropical types. The level of genetic diversity and subpopulation structure were associated with the germplasm origin and post-domestication selection. Compared to temperate lines, the tropical lines had a much higher level of genetic diversity with no significant subpopulation structure identified. Significant variation in LD decay distance (2-100 kb) was found across the genome, chromosomal regions and germplasm groups. The average of LD decay distance (10-100 kb) in the temperate germplasm was two to ten times larger than that in the tropical germplasm (5-10 kb). In conclusion, tropical maize not only host high genetic diversity that can be exploited for future plant breeding, but also show rapid LD decay that provides more opportunity for selection.     

Signatures of soft sweeps across the Dt1 locus underlying determinate growth habit in soya bean [Glycine max (L.) Merr.]

Determinate growth habit is an agronomically important trait associated with domestication in soya bean. Previous studies have demonstrated that the emergence of determinacy is correlated with artificial selection on four nonsynonymous mutations in the Dt1 gene. To better understand the signatures of the soft sweeps across the Dt1 locus and track the origins of the determinate alleles, we examined patterns of nucleotide variation in Dt1 and the surrounding genomic region of approximately 800 kb. Four local, asymmetrical hard sweeps on four determinate alleles, sized approximately 660, 120, 220 and 150 kb, were identified, which constitute the soft sweeps for the adaptation. These variable‐sized sweeps substantially reflected the strength and timing of selection and indicated that the selection on the alleles had been completed rapidly within half a century. Statistics of EHH, iHS, H12 and H2/H1 based on haplotype data had the power to detect the soft sweeps, revealing distinct signatures of extensive long‐range LD and haplotype homozygosity, and multiple frequent adaptive haplotypes. A haplotype network constructed for Dt1 and a phylogenetic tree based on its extended haplotype block implied independent sources of the adaptive alleles through de novo mutations or rare standing variation in quick succession during the selective phase, strongly supporting multiple origins of the determinacy. We propose that the adaptation of soya bean determinacy is guided by a model of soft sweeps and that this model might be indispensable during crop domestication or evolution.     

Excess Polymorphism at the Adh Locus in DROSOPHILA MELANOGASTER

The evolutionary history of a region of DNA encompassing the Adh locus is studied by comparing patterns of variation in Drosophila melanogaster and its sibling species, D. simulans. An unexpectedly high level of silent polymorphism in the Adh coding region relative to the 5' and 3' flanking regions in D. melanogaster is revealed by a populational survey of restriction polymorphism using a four-cutter filter hybridization technique as well as by direct sequence comparisons. In both of these studies, a region of the Adh gene encompassing the three coding exons exhibits a frequency of polymorphism equal to that of a 4-kb 5' flanking region. In contrast, an interspecific sequence comparison shows a two-fold higher level of divergence in the 5' flanking sequence compared to the structural locus. Analysis of the patterns of variation suggest an excess of polymorphism within the D. melanogaster Adh locus, rather than lack of polymorphism in the 5' flanking region. An approach is outlined for testing neutral theory predictions about patterns of variation within and between species. This approach indicates that the observed patterns of variation are incompatible with an infinite site neutral model.     

Regulation of genome stability by TEL1 and MEC1, yeast homologs of the mammalian ATM and ATR genes

DNA sequence surveys of Drosophila melanogaster populations show a strong positive correlation between the recombination rate experienced by a locus and its level of nucleotide polymorphism. In particular, surveys of the fourth chromosome gene ci(D) show greatly reduced levels of nucleotide variation; this observation was originally interpreted in terms of selective sweeps occurring on the nonrecombining fourth chromosome. Subsequent theoretical work has, however, uncovered several other selective processes that can reduce variation. In this study, we revisit the Drosophila fourth chromosome, investigating variation in 5-6 kb of the gene ankyrin in D. melanogaster and D. simulans. Silent nucleotide site diversity is approximately 5 x 10(-4) for both species, consistent with the previous observations of low variation at ci(D). Given the observed frequency spectra at ankyrin, coalescent simulations indicate that reduced diversity in the region is unlikely to be due to a selective sweep alone. We find evidence for recombinational exchange at this locus, and both species appear to be fixed for an insertion of the transposable element HB in an intron of ankyrin.     

KRN4 Controls Quantitative Variation in Maize Kernel Row Number

Kernel row number (KRN) is an important component of yield during the domestication and improvement of maize and controlled by quantitative trait loci (QTL). Here, we fine-mapped a major KRN QTL, KRN4, which can enhance grain productivity by increasing KRN per ear. We found that a ~3-Kb intergenic region about 60 Kb downstream from the SBP-box gene Unbranched3 (UB3) was responsible for quantitative variation in KRN by regulating the level of UB3 expression. Within the 3-Kb region, the 1.2-Kb Presence-Absence variant was found to be strongly associated with quantitative variation in KRN in diverse maize inbred lines, and our results suggest that this 1.2-Kb transposon-containing insertion is likely responsible for increased KRN. A previously identified A/G SNP (S35, also known as Ser220Asn) in UB3 was also found to be significantly associated with KRN in our association-mapping panel. Although no visible genetic effect of S35 alone could be detected in our linkage mapping population, it was found to genetically interact with the 1.2-Kb PAV to modulate KRN. The KRN4 was under strong selection during maize domestication and the favorable allele for the 1.2-Kb PAV and S35 has been significantly enriched in modern maize improvement process. The favorable haplotype (Hap1) of 1.2-Kb-PAV-S35 was selected during temperate maize improvement, but is still rare in tropical and subtropical maize germplasm. The dissection of the KRN4 locus improves our understanding of the genetic basis of quantitative variation in complex traits in maize.     

Identifying loci with breeding potential across temperate and tropical adaptation via EigenGWAS and EnvGWAS

Understanding the genomic basis of adaptation in maize is important for gene discovery and the improvement of breeding germplasm, but much remains a mystery in spite of significant population genetics and archaeological research. Identifying the signals underpinning adaptation are challenging as adaptation often coincided with genetic drift, and the base genomic diversity of the species in massive. In this study, tGBS technology was used to genotype 1,143 diverse maize accessions including landraces collected from 20 countries and elite breeding lines of tropical lowland, highland, subtropical/midaltitude and temperate ecological zones. Based on 355,442 high‐quality single nucleotide polymorphisms, 13 genomic regions were detected as being under selection using the bottom‐up searching strategy, EigenGWAS. Of the 13 selection regions, 10 were first reported, two were associated with environmental parameters via EnvGWAS, and 146 genes were enriched. Combining large‐scale genomic and ecological data in this diverse maize panel, our study supports a polygenic adaptation model of maize and offers a framework to enhance our understanding of both the mechanistic basis and the evolutionary consequences of maize domestication and adaptation. The regions identified here are promising candidates for further, targeted exploration to identify beneficial alleles and haplotypes for deployment in maize breeding.     

Molecular characterization of global maize breeding germplasm based on genome-wide single nucleotide polymorphisms

Characterization of genetic diversity is of great value to assist breeders in parental line selection and breeding system design. We screened 770 maize inbred lines with 1,034 single nucleotide polymorphism (SNP) markers and identified 449 high-quality markers with no germplasm-specific biasing effects. Pairwise comparisons across three distinct sets of germplasm, CIMMYT (394), China (282), and Brazil (94), showed that the elite lines from these diverse breeding pools have been developed with only limited utilization of genetic diversity existing in the center of origin. Temperate and tropical/subtropical germplasm clearly clustered into two separate groups. The temperate germplasm could be further divided into six groups consistent with known heterotic patterns. The greatest genetic divergence was observed between temperate and tropical/subtropical lines, followed by the divergence between yellow and white kernel lines, whereas the least divergence was observed between dent and flint lines. Long-term selection for hybrid performance has contributed to significant allele differentiation between heterotic groups at 20% of the SNP loci. There appeared to be substantial levels of genetic variation between different breeding pools as revealed by missing and unique alleles. Two SNPs developed from the same candidate gene were associated with the divergence between two opposite Chinese heterotic groups. Associated allele frequency change at two SNPs and their allele missing in Brazilian germplasm indicated a linkage disequilibrium block of 142 kb. These results confirm the power of SNP markers for diversity analysis and provide a feasible approach to unique allele discovery and use in maize breeding programs.     

The molecular evolution of terminal ear1, a regulatory gene in the genus Zea.

Nucleotide diversity in the terminal ear1 (te1) gene, a regulatory locus hypothesized to be involved in the morphological evolution of maize (Zea mays ssp. mays), was investigated for evidence of past selection. Nucleotide polymorphism in a 1.4-kb region of te1 was analyzed for a sample of 26 sequences isolated from 12 maize lines, five populations of the maize progenitor, Z. mays ssp. parviglumis, six other Zea populations, and two Tripsacum species. Although nucleotide diversity in te1 in maize is reduced relative to ssp. parviglumis, phylogenetic and statistical analyses of the pattern of polymorphism among these sequences provided no evidence of past selection, indicating that the region of the gene studied was probably not involved in maize evolution. The level of reduction in genetic diversity in te1 in maize relative to its progenitor is comparable to that found in previous reports for isozymes and other neutrally evolving maize genes and is consistent with a genome-wide reduction of genetic diversity resulting from a domestication bottleneck. An estimate of the age (1.2-1.4 million yr) of the maize gene pool based on te1 is roughly consistent with previous estimates based on other neutral genes, but may be biased by the apparently slow synonymous substitution rate at te1.     

Selection under domestication: evidence for a sweep in the rice waxy genomic region

Rice (Oryza sativa) was cultivated by Asian Neolithic farmers >11,000 years ago, and different cultures have selected for divergent starch qualities in the rice grain during and after the domestication process. An intron 1 splice donor site mutation of the Waxy gene is responsible for the absence of amylose in glutinous rice varieties. This mutation appears to have also played an important role in the origin of low amylose, nonglutinous temperate japonica rice varieties, which form a primary component of Northeast Asian cuisines. Waxy DNA sequence analyses indicate that the splice donor mutation is prevalent in temperate japonica rice varieties, but rare or absent in tropical japonica, indica, aus, and aromatic varieties. Sequence analysis across a 500-kb genomic region centered on Waxy reveals patterns consistent with a selective sweep in the temperate japonicas associated with the mutation. The size of the selective sweep (>250 kb) indicates very strong selection in this region, with an inferred selection coefficient that is higher than similar estimates from maize domestication genes or wild species. These findings demonstrate that selection pressures associated with crop domestication regimes can exceed by one to two orders of magnitude those observed for genes under even strong selection in natural systems.     

Molecular evidence for post-domestication selection in the <Emphasis Type="Italic">Waxy</Emphasis> gene of Chinese waxy maize

Waxy maize was first reported in China in 1909 and is mainly used in food production in Asia. The evidence for strong domestication selection in the Waxy locus of rice and a selective sweep around its genomic region make us to wonder whether there has been similar selection in Waxy in glutinous maize. To address this issue, DNA sequences of Waxy, three flanking genes and an unlinked gene (Adh1) of 30 accessions sampled from Chinese waxy maize accessions, including representative landraces and inbred lines, were determined in this study. Sharp reduction of nucleotide diversity and significant neutrality tests (Tajima’s D and Fu and Li’s F*) were observed in the Waxy locus in Chinese waxy maize but not in nonglutinous maize; comparison with the unlinked gene confirmed that this pattern was different to Waxy. Sequence analysis across a 143 kb genomic segment centered on the Waxy locus revealed patterns consistent with a selective sweep in the upstream region of Waxy. The selective sweep detected based on current limited genomic sequences exceeded over 50 kb, indicating strong selection in this or a bigger region. However, No sweep effect was detected in the repetitive downstream region of Waxy. Phylogenetic analysis indicated that Chinese waxy maize was domesticated from the cultivated flint maize (Zea mays ssp. mays) that was introduced from the new world. At least two independent deletions in exon 7 (30 bp) and 10 (15 bp) were identified in the Chinese accessions respectively. These findings demonstrate a similar pattern of domestication selection in the Waxy genomic region in both glutinous maize and rice, suggesting that this pattern in the rise of glutinous phenotype is likely in other cereal crops.     

Maize adaptation to temperate climate: relationship between population structure and polymorphism in the Dwarf8 gene

To investigate the genetic basis of maize adaptation to temperate climate, collections of 375 inbred lines and 275 landraces, representative of American and European diversity, were evaluated for flowering time under short- and long-day conditions. The inbred line collection was genotyped for 55 genomewide simple sequence repeat (SSR) markers. Comparison of inbred line population structure with that of landraces, as determined with 24 SSR loci, underlined strong effects of both historical and modern selection on population structure and a clear relationship with geographical origins. The late tropical groups and the early "Northern Flint" group from the northern United States and northern Europe exhibited different flowering times. Both collections were genotyped for a 6-bp insertion/deletion in the Dwarf8 (D8idp) gene, previously reported to be potentially involved in flowering time variation in a 102 American inbred panel. Among-group D8idp differentiation was much higher than that for any SSR marker, suggesting diversifying selection. Correcting for population structure, D8idp was associated with flowering time under long-day conditions, the deletion allele showing an average earlier flowering of 29 degree days for inbreds and 145 degree days for landraces. Additionally, the deletion allele occurred at a high frequency (>80%) in Northern Flint while being almost absent (<5%) in tropical materials. Altogether, these results indicate that Dwarf8 could be involved in maize climatic adaptation through diversifying selection for flowering time.     

Estimating a nucleotide substitution rate for maize from polymorphism at a major domestication locus

To estimate a rate for single nucleotide substitutions for maize (Zea mays ssp. mays), we have taken advantage of data from genetic and archaeological studies of the domestication of maize from its wild ancestor, teosinte (Z. mays ssp. parviglumis). Genetic studies have shown that the teosinte branched1 (tb1) gene was a major target of human selection during maize domestication, and sequence diversity in the intergenic region 5' to the tb1-coding sequence is extraordinarily low. We show that polymorphism in this region is consistent with new mutation following fixation for a small number of tb1 haplotypes during domestication. Archeological studies suggest that maize was domesticated approximately 6,250-10,000 years ago and subsequently the size of the maize population is thought to have expanded rapidly. Using the observed number of mutations within the region of selection at tb1, the approximate age of maize domestication, and approximations for the maize genealogy, we have derived estimates for the nucleotide substitution rate for the tb1 intergenic region. Using two approaches, one of which is a coalescent approach, we obtain rate estimates of approximately 2.9 x 10(-8) and 3.3 x 10(-8) substitutions per site per year. We also show that the pattern of polymorphism in the tb1 intergenic region appears to have been strongly affected by the mutagenic effect of DNA methylation. Excluding target sites of symmetric DNA methylation (CG and CNG sites) from analysis, the mutation rate estimates are reduced by approximately 50%-60%, while the rates for CG and CNG sites are nearly an order of magnitude higher. We use rate estimates from the tb1 region to estimate the timing of expansion of transposable elements in the maize genome and suggest that this expansion occurred primarily within the last million years.     

Neutral and Non-Neutral Evolution of Drosophila Mitochondrial DNA

To test hypotheses of neutral evolution of mitochondrial DNA (mtDNA), nucleotide sequences were determined for 1515 base pairs of the NADH dehydrogenase subunit 5 (ND5) gene in the mitochondrial DNA of 29 lines of Drosophila melanogaster and 9 lines of its sibling species Drosophila simulans. In contrast to the patterns for nuclear genes, where D. melanogaster generally exhibits much less nucleotide polymorphism, the number of segregating sites was slightly higher in a global sample of nine ND5 sequences in D. melanogaster (s = 8) than in the nine lines of D. simulans (s = 6). When compared to variation at nuclear loci, the mtDNA variation in D. melanogaster does not depart from neutral expectations. The ND5 sequences in D. simulans, however, show fewer than half the number of variable sites expected under neutrality when compared to sequences from the period locus. While this reduction in variation is not significant at the 5% level, HKA tests with published restriction data for mtDNA in D. simulans do show a significant reduction of variation suggesting a selective sweep of variation in the mtDNA in this species. Tests of neutral evolution based on the ratios of synonymous and replacement polymorphism and divergence are generally consistent with neutral expectations, although a significant excess of amino acid polymorphism within both species is localized in one region of the protein. The rate of mtDNA evolution has been faster in D. melanogaster than in D. simulans and the population structure of mtDNA is distinct in these species. The data reveal how different rates of mtDNA evolution between species and different histories of neutral and adaptive evolution within species can compromise historical inferences in population and evolutionary biology.     

Inversion frequencies in Drosophila serrata along an eastern Australian transect.

Clinal patterns over broad geographic regions provide a way of identifying characteristics of species under selection and are increasingly being used in quantitative trait locus mapping of adaptive genetic variation in Drosophila. However, interpretations of clinal patterns can be complicated by inversions that also vary clinally and reduce recombination in some parts of the genome. Drosophila serrata (Malloch) is an Australian endemic species being used to investigate the genetic basis of geographic variation in climatic adaptation and mate recognition. Here we describe inversions in D. serrata populations from the east coast of Australia, covering tropical and temperate regions. Seven autosomal paracentric inversions and 1 apparently complex X chromosome arrangement were identified from these populations. All inverted arrangements were relatively more common in tropical populations; 2 common inversions showed clinal patterns over part of the range of D. serrata. Inversion polymorphism was relatively higher in tropical populations and almost absent in populations near the cooler southern border, in agreement with findings on other Drosophila species. While these patterns will complicate mapping of adaptive variation in D. serrata, they suggest that this species will be useful in investigatingthe dynamics of inversion-trait associations in natural populations.     

Detecting the footprint of positive selection in a european population of Drosophila melanogaster: multilocus pattern of variation and distance to coding regions

The effects on nucleotide variation of adaptations to temperate habitats and of the possible bottleneck associated with the origin of European populations of Drosophila melanogaster should be detectable in DNA sequences given the short time elapsed relative to the species population size. We surveyed nucleotide variation in 109 fragments distributed across the X chromosome in a European population of D. melanogaster to detect the footprint of positive selection. Fragments were located primarily in large noncoding regions. Multilocus tests based on Tajima's D statistic revealed a significant departure from neutral expectations in a stationary panmictic population, with an important contribution from both positive and negative D values. A positive relationship between Tajima's D values and distance to coding region was detected, with a comparative excess of significantly negative D values in the subset of fragments closer to coding regions. Also, there was a significant heterogeneity in the polymorphism to divergence ratio, with 12 fragments contributing 42% to the test statistic. Moreover, these fragments were comparatively closer to coding regions. These findings would imply positive selection events, and thus selective sweeps, during the species expansion to Europe.     

Conditional neutrality at two adjacent NBS-LRR disease resistance loci in natural populations of Arabidopsis lyrata

We examined patterns of nucleotide diversity at a genomic region containing two linked candidate disease resistance (NBS-LRR) genes in seven populations of the outcrossing plant Arabidopsis lyrata. In comparison with two adjacent control genes and neutral reference genes across the genome, the NBS-LRR genes exhibited elevated nonsynonymous variation and a large number of major-effect polymorphisms causing early stop codons and/or frameshift mutations. In contrast, analysis of synonymous diversity provided no evidence that the region was subject to long-term balancing selection or recent selective sweeps in any of the seven populations surveyed. Also in contrast with earlier surveys of one of these R genes, there was no evidence that the resistance genes or the major-effect mutations were subject to elevated differentiation between populations. We suggest that conditional neutrality in the absence of the corresponding pathogen, rather than long-term balancing selection or local adaptation, may in some circumstances be a significant cause of elevated functional polymorphism at R genes. In contrast with the R genes, analysis of diversity and differentiation at the flanking FERONIA locus showed high population divergence, suggesting local adaptation on this locus controlling male-female signalling during fertilization.     

Molecular characterization of the waxy locus in sorghum

A comparison of approximately 4.5 kb of nucleotide sequence from the waxy locus (the granule-bound starch synthase I [GBSS I] locus) from a waxy line, BTxARG1, and a non-waxy line, QL39, revealed an extremely high level of sequence conservation. Among a total of 24 nucleotide differences and 9 indels, only 2 nucleotide changes resulted in altered amino acid residues. Protein folding prediction software suggested that one of the amino acid changes (Glu to His) may result in an altered protein structure, which may explain the apparently inactive GBSS I present in BTxARG1. This SNP was not found in the second waxy line, RTx2907, which does not produce GBSS I, and no other SNPs or indels were found in the approximately 4 kb of sequence obtained from RTx2907. Using one indel, the waxy locus was mapped to sorghum chromosome SBI-10, which is syntenous to maize chromosome 9; the waxy locus has been mapped to this maize chromosome. The distribution of indels in a diverse set of sorghum germplasm suggested that there are two broad types of non-waxy GBSS I alleles, each type comprising several alleles, and that the two waxy alleles in BTxARG1 and RTx2907 have evolved from one of the non-waxy allele types. The Glu/His polymorphism was found only in BTxARG1 and derived lines and has potential as a perfect marker for the BTxARG1 source of the waxy allele at the GBSS I locus. The indels correctly predicted the non-waxy phenotype in approximately 65% of diverse sorghum germplasm. The indels co-segregated perfectly with phenotype in two sorghum populations derived from crosses between a waxy and a non-waxy sorghum line, correctly identifying heterozygous lines. Thus, these indel markers or sequence-based SNP markers can be used to follow waxy alleles in sorghum breeding programs in selected pedigrees.     

Genetic Characterization and Linkage Disequilibrium Estimation of a Global Maize Collection Using SNP Markers

A newly developed maize Illumina GoldenGate Assay with 1536 SNPs from 582 loci was used to genotype a highly diverse global maize collection of 632 inbred lines from temperate, tropical, and subtropical public breeding programs. A total of 1229 informative SNPs and 1749 haplotypes within 327 loci was used to estimate the genetic diversity, population structure, and familial relatedness. Population structure identified tropical and temperate subgroups, and complex familial relationships were identified within the global collection. Linkage disequilibrium (LD) was measured overall and within chromosomes, allelic frequency groups, subgroups related by geographic origin, and subgroups of different sample sizes. The LD decay distance differed among chromosomes and ranged between 1 to 10 kb. The LD distance increased with the increase of minor allelic frequency (MAF), and with smaller sample sizes, encouraging caution when using too few lines in a study. The LD decay distance was much higher in temperate than in tropical and subtropical lines, because tropical and subtropical lines are more diverse and contain more rare alleles than temperate lines. A core set of inbreds was defined based on haplotypes, and 60 lines capture 90% of the haplotype diversity of the entire panel. The defined core sets and the entire collection can be used widely for different research targets.     

Naturally occurring indel variation in the Brassica nigra COL1 gene is associated with variation in flowering time

Previous QTL mapping identified a Brassica nigra homolog to Arabidopsis thaliana CO as a candidate gene affecting flowering time in B. nigra. Transformation of an A. thaliana co mutant with two different alleles of the B. nigra CO (Bni COa) homolog, one from an early-flowering B. nigra plant and one from a late one, did not show any differential effect of the two alleles on flowering time. The DNA sequence of the coding region of the two alleles was also identical, showing that nucleotide variation influencing flowering time must reside outside the coding region of Bni COa. In contrast, the nucleotide sequence of the B. nigra COL1 (Bni COL1) gene located 3.5 kb upstream of Bni COa was highly diverged between the alleles from early and late plants. One indel polymorphism in the Bni COL1 coding region, present in several natural populations of B. nigra, displayed a significant association with flowering time within a majority of these populations. These data indicate that a quantitative trait nucleotide (QTN) affecting flowering time is located within or close to the Bni COL1 gene. The intergenic sequence between Bni COL1 and Bni COa displayed a prominent peak of divergence 1 kb downstream of the Bni COL1 coding region. This region could contain regulatory elements for the downstream Bni COa gene. Our data suggest that a naturally occurring QTN for flowering time affects the function or expression of either Bni COL1 or Bni COa.