A genetic tradeoff for tolerance to moderate and severe heat stress in US hybrid maize.

Global climate change is increasing both average temperatures and the frequencies of extreme high temperatures. Past studies have documented a strong negative effect of exposures to temperatures >30°C on hybrid maize yields. However, these studies could not disentangle genetic adaptation via artificial selection from changes in agronomic practices. Because most of the earliest maize hybrids are no longer available, side-by-side comparisons with modern hybrids under current field conditions are generally impossible. Here, we report on the collection and curation of 81 years of public yield trial records covering 4,730 maize hybrids, which enabled us to model genetic variation for temperature responses among maize hybrids. We show that selection may have indirectly and inconsistently contributed to the genetic adaptation of maize to moderate heat stress over this time period while preserving genetic variance for continued adaptation. However, our results reveal the existence of a genetic tradeoff for tolerance to moderate and severe heat stress, leading to a decrease in tolerance to severe heat stress over the same time period. Both trends are particularly conspicuous since the mid-1970s. Such a tradeoff poses challenges to the continued adaptation of maize to warming climates due to a projected increase in the frequency of extreme heat events. Nevertheless, given recent advances in phenomics, enviromics, and physiological modeling, our results offer a degree of optimism for the capacity of plant breeders to adapt maize to warming climates, assuming appropriate levels of R&D investment.

Co-expression analysis aids in the identification of genes in the cuticular wax pathway in maize.

Epicuticular waxes provide a hydrophobic barrier that protects land plants from environmental stresses. To elucidate the molecular functions of maize glossy mutants that reduce the accumulation of epicuticular waxes, eight non-allelic glossy mutants were subjected to transcriptomic comparisons with their respective wild-type siblings. Transcriptomic comparisons identified 2279 differentially expressed (DE) genes. Other glossy genes tended to be down-regulated in glossy mutants; by contrast stress-responsive pathways were induced in mutants. Gene co-expression network (GCN) analysis found that glossy genes were clustered, suggestive of co-regulation. Genes that potentially regulate the accumulation of glossy gene transcripts were identified via a pathway level co-expression analysis. Expression data from diverse organs showed that maize glossy genes are generally active in young leaves, silks, and tassels, while largely inactive in seeds and roots. Through reverse genetics, a DE gene homologous to Arabidopsis CER8 and co-expressed with known glossy genes was confirmed to participate in epicuticular wax accumulation. GCN data-informed forward genetics approach enabled cloning of the gl14 gene, which encodes a putative membrane-associated protein. Our results deepen understanding of the transcriptional regulation of the genes involved in the accumulation of epicuticular wax, and provide two maize glossy genes and a number of candidate genes for further characterization.

Intragenic Meiotic Crossovers Generate Novel Alleles with Transgressive Expression Levels.

Meiotic recombination is an evolutionary force that generates new genetic diversity upon which selection can act. Whereas multiple studies have assessed genome-wide patterns of recombination and specific cases of intragenic recombination, few studies have assessed intragenic recombination genome-wide in higher eukaryotes. We identified recombination events within or near genes in a population of maize recombinant inbred lines (RILs) using RNA-sequencing data. Our results are consistent with case studies that have shown that intragenic crossovers cluster at the 5' ends of some genes. Further, we identified cases of intragenic crossovers that generate transgressive transcript accumulation patterns, that is, recombinant alleles displayed higher or lower levels of expression than did nonrecombinant alleles in any of ∼100 RILs, implicating intragenic recombination in the generation of new variants upon which selection can act. Thousands of apparent gene conversion events were identified, allowing us to estimate the genome-wide rate of gene conversion at SNP sites (4.9 × 10-5). The density of syntenic genes (i.e., those conserved at the same genomic locations since the divergence of maize and sorghum) exhibits a substantial correlation with crossover frequency, whereas the density of nonsyntenic genes (i.e., those which have transposed or been lost subsequent to the divergence of maize and sorghum) shows little correlation, suggesting that crossovers occur at higher rates in syntenic genes than in nonsyntenic genes. Increased rates of crossovers in syntenic genes could be either a consequence of the evolutionary conservation of synteny or a biological process that helps to maintain synteny.

tGBS® genotyping-by-sequencing enables reliable genotyping of heterozygous loci.

Conventional genotyping-by-sequencing (cGBS) strategies suffer from high rates of missing data and genotyping errors, particularly at heterozygous sites. tGBS® genotyping-by-sequencing is a novel method of genome reduction that employs two restriction enzymes to generate overhangs in opposite orientations to which (single-strand) oligos rather than (double-stranded) adaptors are ligated. This strategy ensures that only double-digested fragments are amplified and sequenced. The use of oligos avoids the necessity of preparing adaptors and the problems associated with inter-adaptor annealing/ligation. Hence, the tGBS protocol simplifies the preparation of high-quality GBS sequencing libraries. During polymerase chain reaction (PCR) amplification, selective nucleotides included at the 3'-end of the PCR primers result in additional genome reduction as compared to cGBS. By adjusting the number of selective bases, different numbers of genomic sites are targeted for sequencing. Therefore, for equivalent amounts of sequencing, more reads per site are available for SNP calling. Hence, as compared to cGBS, tGBS delivers higher SNP calling accuracy (>97-99%), even at heterozygous sites, less missing data per marker across a population of samples, and an enhanced ability to genotype rare alleles. tGBS is particularly well suited for genomic selection, which often requires the ability to genotype populations of individuals that are heterozygous at many loci.

Linked read technology for assembling large complex and polyploid genomes.

BACKGROUND: Short read DNA sequencing technologies have revolutionized genome assembly by providing high accuracy and throughput data at low cost. But it remains challenging to assemble short read data, particularly for large, complex and polyploid genomes. The linked read strategy has the potential to enhance the value of short reads for genome assembly because all reads originating from a single long molecule of DNA share a common barcode. However, the majority of studies to date that have employed linked reads were focused on human haplotype phasing and genome assembly. RESULTS: Here we describe a de novo maize B73 genome assembly generated via linked read technology which contains ~ 172,000 scaffolds with an N50 of 89 kb that cover 50% of the genome. Based on comparisons to the B73 reference genome, 91% of linked read contigs are accurately assembled. Because it was possible to identify errors with > 76% accuracy using machine learning, it may be possible to identify and potentially correct systematic errors. Complex polyploids represent one of the last grand challenges in genome assembly. Linked read technology was able to successfully resolve the two subgenomes of the recent allopolyploid, proso millet (Panicum miliaceum). Our assembly covers ~ 83% of the 1 Gb genome and consists of 30,819 scaffolds with an N50 of 912 kb. CONCLUSIONS: Our analysis provides a framework for future de novo genome assemblies using linked reads, and we suggest computational strategies that if implemented have the potential to further improve linked read assemblies, particularly for repetitive genomes.

Nonsyntenic genes drive highly dynamic complementation of gene expression in maize hybrids.

Maize (Zea mays) displays an exceptional level of structural genomic diversity, which is likely unique among higher eukaryotes. In this study, we surveyed how the genetic divergence of two maize inbred lines affects the transcriptomic landscape in four different primary root tissues of their F1-hybrid progeny. An extreme instance of complementation was frequently observed: genes that were expressed in only one parent but in both reciprocal hybrids. This single-parent expression (SPE) pattern was detected for 2341 genes with up to 1287 SPE patterns per tissue. As a consequence, the number of active genes in hybrids exceeded that of their parents in each tissue by >400. SPE patterns are highly dynamic, as illustrated by their excessive degree of tissue specificity (80%). The biological significance of this type of complementation is underpinned by the observation that a disproportionally high number of SPE genes (75 to 82%) is nonsyntenic, as opposed to all expressed genes (36%). These genes likely evolved after the last whole-genome duplication and are therefore younger than the syntenic genes. In summary, SPE genes shape the remarkable gene expression plasticity between root tissues and complementation in maize hybrids, resulting in a tissue-specific increase of active genes in F1-hybrids compared with their inbred parents.

Extreme-phenotype genome-wide association study (XP-GWAS): a method for identifying trait-associated variants by sequencing pools of individuals selected from a diversity panel.

Although approaches for performing genome-wide association studies (GWAS) are well developed, conventional GWAS requires high-density genotyping of large numbers of individuals from a diversity panel. Here we report a method for performing GWAS that does not require genotyping of large numbers of individuals. Instead XP-GWAS (extreme-phenotype GWAS) relies on genotyping pools of individuals from a diversity panel that have extreme phenotypes. This analysis measures allele frequencies in the extreme pools, enabling discovery of associations between genetic variants and traits of interest. This method was evaluated in maize (Zea mays) using the well-characterized kernel row number trait, which was selected to enable comparisons between the results of XP-GWAS and conventional GWAS. An exome-sequencing strategy was used to focus sequencing resources on genes and their flanking regions. A total of 0.94 million variants were identified and served as evaluation markers; comparisons among pools showed that 145 of these variants were statistically associated with the kernel row number phenotype. These trait-associated variants were significantly enriched in regions identified by conventional GWAS. XP-GWAS was able to resolve several linked QTL and detect trait-associated variants within a single gene under a QTL peak. XP-GWAS is expected to be particularly valuable for detecting genes or alleles responsible for quantitative variation in species for which extensive genotyping resources are not available, such as wild progenitors of crops, orphan crops, and other poorly characterized species such as those of ecological interest.

The maize brown midrib4 (bm4) gene encodes a functional folylpolyglutamate synthase.

Mutations in the brown midrib4 (bm4) gene affect the accumulation and composition of lignin in maize. Fine-mapping analysis of bm4 narrowed the candidate region to an approximately 105 kb interval on chromosome 9 containing six genes. Only one of these six genes, GRMZM2G393334, showed decreased expression in mutants. At least four of 10 Mu-induced bm4 mutant alleles contain a Mu insertion in the GRMZM2G393334 gene. Based on these results, we concluded that GRMZM2G393334 is the bm4 gene. GRMZM2G393334 encodes a putative folylpolyglutamate synthase (FPGS), which functions in one-carbon (C1) metabolism to polyglutamylate substrates of folate-dependent enzymes. Yeast complementation experiments demonstrated that expression of the maize bm4 gene in FPGS-deficient met7 yeast is able to rescue the yeast mutant phenotype, thus demonstrating that bm4 encodes a functional FPGS. Consistent with earlier studies, bm4 mutants exhibit a modest decrease in lignin concentration and an overall increase in the S:G lignin ratio relative to wild-type. Orthologs of bm4 include at least one paralogous gene in maize and various homologs in other grasses and dicots. Discovery of the gene underlying the bm4 maize phenotype illustrates a role for FPGS in lignin biosynthesis.

The maize brown midrib2 (bm2) gene encodes a methylenetetrahydrofolate reductase that contributes to lignin accumulation.

The midribs of maize brown midrib (bm) mutants exhibit a reddish-brown color associated with reductions in lignin concentration and alterations in lignin composition. Here, we report the mapping, cloning, and functional and biochemical analyses of the bm2 gene. The bm2 gene was mapped to a small region of chromosome 1 that contains a putative methylenetetrahydrofolate reductase (MTHFR) gene, which is down-regulated in bm2 mutant plants. Analyses of multiple Mu-induced bm2-Mu mutant alleles confirmed that this constitutively expressed gene is bm2. Yeast complementation experiments and a previously published biochemical characterization show that the bm2 gene encodes a functional MTHFR. Quantitative RT-PCR analyses demonstrated that the bm2 mutants accumulate substantially reduced levels of bm2 transcript. Alteration of MTHFR function is expected to influence accumulation of the methyl donor S-adenosyl-L-methionine (SAM). Because SAM is consumed by two methyltransferases in the lignin pathway (Ye et al., ), the finding that bm2 encodes a functional MTHFR is consistent with its lignin phenotype. Consistent with this functional assignment of bm2, the expression patterns of genes in a variety of SAM-dependent or -related pathways, including lignin biosynthesis, are altered in the bm2 mutant. Biochemical assays confirmed that bm2 mutants accumulate reduced levels of lignin with altered composition compared to wild-type. Hence, this study demonstrates a role for MTHFR in lignin biosynthesis.

Complementation contributes to transcriptome complexity in maize (Zea mays L.) hybrids relative to their inbred parents.

Typically, F(1)-hybrids are more vigorous than their homozygous, genetically distinct parents, a phenomenon known as heterosis. In the present study, the transcriptomes of the reciprocal maize (Zea mays L.) hybrids B73×Mo17 and Mo17×B73 and their parental inbred lines B73 and Mo17 were surveyed in primary roots, early in the developmental manifestation of heterotic root traits. The application of statistical methods and a suitable experimental design established that 34,233 (i.e., 86%) of all high-confidence maize genes were expressed in at least one genotype. Nearly 70% of all expressed genes were differentially expressed between the two parents and 42%-55% of expressed genes were differentially expressed between one of the parents and one of the hybrids. In both hybrids, ∼10% of expressed genes exhibited nonadditive gene expression. Consistent with the dominance model (i.e., complementation) for heterosis, 1124 genes that were expressed in the hybrids were expressed in only one of the two parents. For 65 genes, it could be shown that this was a consequence of complementation of genomic presence/absence variation. For dozens of other genes, alleles from the inactive inbred were activated in the hybrid, presumably via interactions with regulatory factors from the active inbred. As a consequence of these types of complementation, both hybrids expressed more genes than did either parental inbred. Finally, in hybrids, ∼14% of expressed genes exhibited allele-specific expression (ASE) levels that differed significantly from the parental-inbred expression ratios, providing further evidence for interactions of regulatory factors from one parental genome with target genes from the other parental genome.

Genic and nongenic contributions to natural variation of quantitative traits in maize.

The complex genomes of many economically important crops present tremendous challenges to understand the genetic control of many quantitative traits with great importance in crop production, adaptation, and evolution. Advances in genomic technology need to be integrated with strategic genetic design and novel perspectives to break new ground. Complementary to individual-gene-targeted research, which remains challenging, a global assessment of the genomic distribution of trait-associated SNPs (TASs) discovered from genome scans of quantitative traits can provide insights into the genetic architecture and contribute to the design of future studies. Here we report the first systematic tabulation of the relative contribution of different genomic regions to quantitative trait variation in maize. We found that TASs were enriched in the nongenic regions, particularly within a 5-kb window upstream of genes, which highlights the importance of polymorphisms regulating gene expression in shaping the natural variation. Consistent with these findings, TASs collectively explained 44%-59% of the total phenotypic variation across maize quantitative traits, and on average, 79% of the explained variation could be attributed to TASs located in genes or within 5 kb upstream of genes, which together comprise only 13% of the genome. Our findings suggest that efficient, cost-effective genome-wide association studies (GWAS) in species with complex genomes can focus on genic and promoter regions.

Spreading of heterochromatin is limited to specific families of maize retrotransposons.

Transposable elements (TEs) have the potential to act as controlling elements to influence the expression of genes and are often subject to heterochromatic silencing. The current paradigm suggests that heterochromatic silencing can spread beyond the borders of TEs and influence the chromatin state of neighboring low-copy sequences. This would allow TEs to condition obligatory or facilitated epialleles and act as controlling elements. The maize genome contains numerous families of class I TEs (retrotransposons) that are present in moderate to high copy numbers, and many are found in regions near genes, which provides an opportunity to test whether the spreading of heterochromatin from retrotransposons is prevalent. We have investigated the extent of heterochromatin spreading into DNA flanking each family of retrotransposons by profiling DNA methylation and di-methylation of lysine 9 of histone 3 (H3K9me2) in low-copy regions of the maize genome. The effects of different retrotransposon families on local chromatin are highly variable. Some retrotransposon families exhibit enrichment of heterochromatic marks within 800-1,200 base pairs of insertion sites, while other families exhibit very little evidence for the spreading of heterochromatic marks. The analysis of chromatin state in genotypes that lack specific insertions suggests that the heterochromatin in low-copy DNA flanking retrotransposons often results from the spreading of silencing marks rather than insertion-site preferences. Genes located near TEs that exhibit spreading of heterochromatin tend to be expressed at lower levels than other genes. Our findings suggest that a subset of retrotransposon families may act as controlling elements influencing neighboring sequences, while the majority of retrotransposons have little effect on flanking sequences.

The Aux/IAA gene rum1 involved in seminal and lateral root formation controls vascular patterning in maize (Zea mays L.) primary roots.

The maize (Zea mays L.) Aux/IAA protein RUM1 (ROOTLESS WITH UNDETECTABLE MERISTEMS 1) controls seminal and lateral root initiation. To identify RUM1-dependent gene expression patterns, RNA-Seq of the differentiation zone of primary roots of rum1 mutants and the wild type was performed in four biological replicates. In total, 2 801 high-confidence maize genes displayed differential gene expression with Fc ≥2 and FDR ≤1%. The auxin signalling-related genes rum1, like-auxin1 (lax1), lax2, (nam ataf cuc 1 nac1), the plethora genes plt1 (plethora 1), bbm1 (baby boom 1), and hscf1 (heat shock complementing factor 1) and the auxin response factors arf8 and arf37 were down-regulated in the mutant rum1. All of these genes except nac1 were auxin-inducible. The maize arf8 and arf37 genes are orthologues of Arabidopsis MP/ARF5 (MONOPTEROS/ARF5), which controls the differentiation of vascular cells. Histological analyses of mutant rum1 roots revealed defects in xylem organization and the differentiation of pith cells around the xylem. Moreover, histochemical staining of enlarged pith cells surrounding late metaxylem elements demonstrated that their thickened cell walls displayed excessive lignin deposition. In line with this phenotype, rum1-dependent mis-expression of several lignin biosynthesis genes was observed. In summary, RNA-Seq of RUM1-dependent gene expression in maize primary roots, in combination with histological and histochemical analyses, revealed the specific regulation of auxin signal transduction components by RUM1 and novel functions of RUM1 in vascular development.

Parent-of-origin effects on gene expression and DNA methylation in the maize endosperm.

Imprinting describes the differential expression of alleles based on their parent of origin. Deep sequencing of RNAs from maize (Zea mays) endosperm and embryo tissue 14 d after pollination was used to identify imprinted genes among a set of ~12,000 genes that were expressed and contained sequence polymorphisms between the B73 and Mo17 genotypes. The analysis of parent-of-origin patterns of expression resulted in the identification of 100 putative imprinted genes in maize endosperm, including 54 maternally expressed genes (MEGs) and 46 paternally expressed genes (PEGs). Three of these genes have been previously identified as imprinted, while the remaining 97 genes represent novel imprinted maize genes. A genome-wide analysis of DNA methylation identified regions with reduced endosperm DNA methylation in, or near, 19 of the 100 imprinted genes. The reduced levels of DNA methylation in endosperm are caused by hypomethylation of the maternal allele for both MEGs and PEGs in all cases tested. Many of the imprinted genes with reduced DNA methylation levels also show endosperm-specific expression patterns. The imprinted maize genes were compared with imprinted genes identified in genome-wide screens of rice (Oryza sativa) and Arabidopsis thaliana, and at least 10 examples of conserved imprinting between maize and each of the other species were identified.

Heritable epigenetic variation among maize inbreds.

Epigenetic variation describes heritable differences that are not attributable to changes in DNA sequence. There is the potential for pure epigenetic variation that occurs in the absence of any genetic change or for more complex situations that involve both genetic and epigenetic differences. Methylation of cytosine residues provides one mechanism for the inheritance of epigenetic information. A genome-wide profiling of DNA methylation in two different genotypes of Zea mays (ssp. mays), an organism with a complex genome of interspersed genes and repetitive elements, allowed the identification and characterization of examples of natural epigenetic variation. The distribution of DNA methylation was profiled using immunoprecipitation of methylated DNA followed by hybridization to a high-density tiling microarray. The comparison of the DNA methylation levels in the two genotypes, B73 and Mo17, allowed for the identification of approximately 700 differentially methylated regions (DMRs). Several of these DMRs occur in genomic regions that are apparently identical by descent in B73 and Mo17 suggesting that they may be examples of pure epigenetic variation. The methylation levels of the DMRs were further studied in a panel of near-isogenic lines to evaluate the stable inheritance of the methylation levels and to assess the contribution of cis- and trans- acting information to natural epigenetic variation. The majority of DMRs that occur in genomic regions without genetic variation are controlled by cis-acting differences and exhibit relatively stable inheritance. This study provides evidence for naturally occurring epigenetic variation in maize, including examples of pure epigenetic variation that is not conditioned by genetic differences. The epigenetic differences are variable within maize populations and exhibit relatively stable trans-generational inheritance. The detected examples of epigenetic variation, including some without tightly linked genetic variation, may contribute to complex trait variation.

Changes in genome content generated via segregation of non-allelic homologs.

A careful analysis of two maize recombinant inbred lines (RILs) relative to their inbred parents revealed the presence of several hundred apparently de novo copy number variants (CNVs). These changes in genome content were validated via both PCR and whole exome-array capture-and-sequencing experiments. One hundred and eighty-five genomic regions, which overlap with 38 high-confidence genes, exhibited apparently de novo copy number variation (CNV) in these two RILs and in many instances the same apparently de novo CNV events were observed in multiple RILs. Further analyses revealed that these recurrent apparently de novo CNVs were caused by segregation of single-copy homologous sequences that are located in non-allelic positions in the two parental inbred lines. F(1) individuals derived from these inbred lines will be hemizygous for each of these non-allelic homologs but RIL genotypes will contain these sequences at zero, one or two genomic loci. Hence, the segregation of non-allelic homologs may contribute to transgressive segregation. Indeed, statistical associations between phenotypic quantitative trait loci and genomic losses were observed for two of 14 tested pairs of non-allelic homologs.

B73-Mo17 near-isogenic lines demonstrate dispersed structural variation in maize.

Recombinant inbred lines developed from the maize (Zea mays ssp. mays) inbreds B73 and Mo17 have been widely used to discover quantitative trait loci controlling a wide variety of phenotypic traits and as a resource to produce high-resolution genetic maps. These two parents were used to produce a set of near-isogenic lines (NILs) with small regions of introgression into both backgrounds. A novel array-based genotyping platform was used to score genotypes of over 7,000 loci in 100 NILs with B73 as the recurrent parent and 50 NILs with Mo17 as the recurrent parent. This population contains introgressions that cover the majority of the maize genome. The set of NILs displayed an excess of residual heterozygosity relative to the amount expected based on their pedigrees, and this excess residual heterozygosity is enriched in the low-recombination regions near the centromeres. The genotyping platform provided the ability to survey copy number variants that exist in more copies in Mo17 than in B73. The majority of these Mo17-specific duplications are located in unlinked positions throughout the genome. The utility of this population for the discovery and validation of quantitative trait loci was assessed through analysis of plant height variation.

Mu transposon insertion sites and meiotic recombination events co-localize with epigenetic marks for open chromatin across the maize genome.

The Mu transposon system of maize is highly active, with each of the approximately 50-100 copies transposing on average once each generation. The approximately one dozen distinct Mu transposons contain highly similar approximately 215 bp terminal inverted repeats (TIRs) and generate 9-bp target site duplications (TSDs) upon insertion. Using a novel genome walking strategy that uses these conserved TIRs as primer binding sites, Mu insertion sites were amplified from Mu stocks and sequenced via 454 technology. 94% of approximately 965,000 reads carried Mu TIRs, demonstrating the specificity of this strategy. Among these TIRs, 21 novel Mu TIRs were discovered, revealing additional complexity of the Mu transposon system. The distribution of >40,000 non-redundant Mu insertion sites was strikingly non-uniform, such that rates increased in proportion to distance from the centromere. An identified putative Mu transposase binding consensus site does not explain this non-uniformity. An integrated genetic map containing more than 10,000 genetic markers was constructed and aligned to the sequence of the maize reference genome. Recombination rates (cM/Mb) are also strikingly non-uniform, with rates increasing in proportion to distance from the centromere. Mu insertion site frequencies are strongly correlated with recombination rates. Gene density does not fully explain the chromosomal distribution of Mu insertion and recombination sites, because pronounced preferences for the distal portion of chromosome are still observed even after accounting for gene density. The similarity of the distributions of Mu insertions and meiotic recombination sites suggests that common features, such as chromatin structure, are involved in site selection for both Mu insertion and meiotic recombination. The finding that Mu insertions and meiotic recombination sites both concentrate in genomic regions marked with epigenetic marks of open chromatin provides support for the hypothesis that open chromatin enhances rates of both Mu insertion and meiotic recombination.

Maize inbreds exhibit high levels of copy number variation (CNV) and presence/absence variation (PAV) in genome content.

Following the domestication of maize over the past approximately 10,000 years, breeders have exploited the extensive genetic diversity of this species to mold its phenotype to meet human needs. The extent of structural variation, including copy number variation (CNV) and presence/absence variation (PAV), which are thought to contribute to the extraordinary phenotypic diversity and plasticity of this important crop, have not been elucidated. Whole-genome, array-based, comparative genomic hybridization (CGH) revealed a level of structural diversity between the inbred lines B73 and Mo17 that is unprecedented among higher eukaryotes. A detailed analysis of altered segments of DNA conservatively estimates that there are several hundred CNV sequences among the two genotypes, as well as several thousand PAV sequences that are present in B73 but not Mo17. Haplotype-specific PAVs contain hundreds of single-copy, expressed genes that may contribute to heterosis and to the extraordinary phenotypic diversity of this important crop.

Detailed analysis of a contiguous 22-Mb region of the maize genome.

Most of our understanding of plant genome structure and evolution has come from the careful annotation of small (e.g., 100 kb) sequenced genomic regions or from automated annotation of complete genome sequences. Here, we sequenced and carefully annotated a contiguous 22 Mb region of maize chromosome 4 using an improved pseudomolecule for annotation. The sequence segment was comprehensively ordered, oriented, and confirmed using the maize optical map. Nearly 84% of the sequence is composed of transposable elements (TEs) that are mostly nested within each other, of which most families are low-copy. We identified 544 gene models using multiple levels of evidence, as well as five miRNA genes. Gene fragments, many captured by TEs, are prevalent within this region. Elimination of gene redundancy from a tetraploid maize ancestor that originated a few million years ago is responsible in this region for most disruptions of synteny with sorghum and rice. Consistent with other sub-genomic analyses in maize, small RNA mapping showed that many small RNAs match TEs and that most TEs match small RNAs. These results, performed on approximately 1% of the maize genome, demonstrate the feasibility of refining the B73 RefGen_v1 genome assembly by incorporating optical map, high-resolution genetic map, and comparative genomic data sets. Such improvements, along with those of gene and repeat annotation, will serve to promote future functional genomic and phylogenomic research in maize and other grasses.