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`Review
`Allelic variation and heterosis in maize: How do two
`halves make more than a whole?
`Nathan M. Springer1 and Robert M. Stupar
`Cargill Center for Microbial and Plant Genomics, Department of Plant Biology, University of Minnesota, Saint Paul,
`Minnesota 55108, USA
`
`In this review, we discuss the recent research on allelic variation in maize and possible implications of this work
`toward our understanding of heterosis. Heterosis, or hybrid vigor, is the increased performance of a hybrid relative
`to the parents, and is a result of the variation that is present within a species. Intraspecific comparisons of sequence
`and expression levels in maize have documented a surprisingly high level of allelic variation, which includes variation
`for the content of genic fragments, variation in repetitive elements surrounding genes, and variation in gene
`expression levels. There is evidence that transposons and repetitive DNA play a major role in the generation of this
`allelic diversity. The combination of allelic variants provides a more comprehensive suite of alleles in the hybrid that
`may be involved in novel allelic interactions. A major unresolved question is how the combined allelic variation and
`interactions in a hybrid give rise to heterotic phenotypes. An understanding of allelic variation present in maize
`provides an opportunity to speculate on mechanisms that might lead to heterosis. Variation for the presence of
`genes, the presence of novel beneficial alleles, and modified levels of gene expression in hybrids may all contribute
`to the heterotic phenotypes.
`
`It is often argued that a basic understanding of a process is critical
`to its use and manipulation. Arguably, one of the most notable
`exceptions to this idea is the phenomenon of heterosis. Hetero-
`sis, or hybrid vigor, has been the subject of intense research and
`speculation for well over a century; however, the basic mecha-
`nisms that cause or contribute to heterosis remain unclear (Coors
`and Pandey 1999). Despite this lack of understanding, breeders
`have quite successfully manipulated heterosis to increase the
`vigor of many domesticated species. In maize, it is estimated that
`the use of hybrids and heterosis increases yields ∼15% per annum
`(Duvick 1999).
`Heterosis refers to the phenomenon in which the hybrid F1
`offspring exhibit phenotypic characteristics that are superior to
`the mean of the two parents (mid-parent heterosis), or the better
`of the two parents (better parent heterosis) (Fig. 1). While mid-
`parent heterosis is scientifically interesting, it has relatively little
`economic importance. Better parent heterosis is the underlying
`rationale for the widespread use of hybrids in many agricultural
`species, and we focus our discussion on this phenomenon. The
`level of heterosis can be quantified for specific traits. “Heterosis
`per se” for a specific trait is quantified as the phenotype of the
`hybrid minus the phenotype of the better parent; subsequently,
`the “percentage of heterosis” is calculated as the heterosis per se
`divided by the better of the parental phenotypes. Often, the par-
`ents of heterotic offspring are inbred. In this case, the quantifi-
`cation of heterosis reflects both hybrid vigor and a recovery from
`inbreeding depression.
`Heterosis has been used in the breeding and production of
`many crop and animal species (Janick 1998; Melchinger and
`Gumber 1998). An early application of heterosis was breeding
`mules, which are derived from crossing a female horse and a male
`donkey (Goldman 1998). As pointed out by Troyer (2006), there
`
`1Corresponding author.
`E-mail springer@umn.edu; fax (612) 625-1738.
`Article published online before print. Article and publication date are at http://
`www.genome.org/cgi/doi/10.1101/gr.5347007.
`
`are useful similarities in the examples of mules and hybrid corn.
`Both mules and hybrid corn exhibit superior phenotypes and
`stress tolerance relative to their parents. Additionally, farmers
`showed a willingness to purchase hybrid corn and mules despite
`obvious drawbacks: these products are added expenses and nei-
`ther produce useful offspring, thus requiring the farmer to pur-
`chase a new organism each generation.
`It is important to note that the application and use of hy-
`brids in different species are influenced by several factors. For
`instance, if the degree of heterosis is relatively low in a given
`species, then it may not be cost-effective to use hybrids for com-
`mercial production. The efficient development of hybrids may be
`limited by the species mating system, as self-pollination and/or
`controlled crosses are difficult or impossible to conduct in many
`species. An efficient F1 hybrid production method is essential for
`hybrid commercialization. In fact, the early maize inbred varie-
`ties had extremely low seed yield, leading to the use of double
`cross hybrids until inbred varieties with higher seed yield could
`be developed (Duvick 2001).
`Heterosis is evident not only in artificially selected popula-
`tions, but also may be observed in natural populations (Mitton
`1998; Hansson and Westerberg 2002). Allelic frequencies in a
`random sample of coniferous trees were in agreement with
`Hardy-Weinberg expectations; however, an excess of heterozy-
`gotes was observed when only the mature, oldest, or largest trees
`were sampled (Mitton and Jeffers 1989). Furthermore, a study of
`Pinyon pines suggested that heterozygotes are more resistant to
`herbivory pressure (Mopper et al. 1991). The exact mechanism
`that leads to the enhanced performance of the heterozygotes in
`these tree species has not been determined, but it is possible that
`heterosis is an important factor in fitness for many organisms.
`Maize provides an excellent system for the study and appli-
`cation of heterosis. A wide range of natural genetic diversity has
`been captured in the current maize germplasm (Flint-Garcia et al.
`2005; Wright et al. 2005; Troyer 2006). Maize is relatively easy to
`self- or cross-pollinate, which has enabled the development of
`both diverse inbreds and many hybrids for evaluation. Much of
`
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`Allelic variation and heterosis
`
`traits in maize and rice (Stuber et al. 1992; Xiao et al. 1995; Li et
`al. 2001; Luo et al. 2001; Lu et al. 2003).
`In the simplest view, the reverse of heterosis is inbreeding
`depression, in which progressive self-pollination or sibling mat-
`ings reduce the genome-wide heterozygosity and overall fitness
`of an organism. Inbreeding depression is likely caused by the
`fixation of deleterious alleles within a lineage. Plant breeders
`have created an “unnatural” inbred state in maize, a naturally
`outcrossing species, which may help explain why maize exhibits
`relatively high heterosis when compared to other species. The
`inbreeding depression in inbred maize lines is alleviated when a
`hybrid is formed, and the plants exhibit superior characteristics.
`A fundamental question is whether the reversal of the effects of
`inbreeding in the F1 hybrid (via complementation of deleterious
`alleles with “good” alleles from the other parent) is sufficient to
`explain heterosis, or whether other mechanisms are at work. Two
`hypotheses, dominance and overdominance, have been pro-
`posed to explain heterosis (Text Box 1).
`In this review, we focus on three key questions that address
`heterosis from a genomics perspective: (1) What allelic differ-
`ences exist between heterotic parental lines in terms of types of
`variation and prevalence of variation? (2) How do these allelic
`variants interact in the F1 hybrid organism? (3) How does the
`interaction of allelic variants result in heterotic phenotypes?
`The term “allelic variation” (broadly defined here as the
`sequence or regulatory differences found in different parental
`genotypes) is used in each of the three questions. While we dis-
`cuss each of these questions in the context of maize, it is antici-
`pated that similar mechanisms apply to other organisms.
`
`Allelic variation in maize
`
`The current era of genomic sequencing and global gene expres-
`sion analysis has provided a wealth of information about intra-
`specific allelic variation. Intraspecific allelic variation can include
`sequence changes, structural changes in the genome, altered ex-
`pression levels, and epigenetic changes. At some level, heterosis
`is the result of variation between the parental lines, since in the
`
`Table 1. Phenotypes and heterosis in B73 relative to Mo17
`
`Trait
`
`B73
`
`Mo17 Hybrid
`
`Yield (mg/ha)b
`Seed numberc
`Seed weight
`(g/ear)d
`Height (cm)d
`Total nodesd
`Ear noded
`Leaf length (cm)d
`Leaf width (mm)d
`Days to first silkd
`Days to first
`antherd
`Tassel branchesd
`Ear length (cm)d
`11-d seedling
`height (cm)c
`11-d seedling
`biomass (g)c
`
`6.32
`430
`
`4.06
`271
`
`10.41
`628
`
`115
`173.3
`14.6
`8.9
`75.3
`90.1
`70.1
`
`68.9
`9.3
`12.7
`
`32.4
`
`98
`169
`13.3
`8.4
`62.7
`84.2
`77.1
`
`71.1
`6.9
`14.3
`
`27.3
`
`232
`211.1
`15.1
`9.2
`87
`106.4
`68.05
`
`65.95
`9.75
`21.2
`
`41
`
`0.42
`
`0.32
`
`0.71
`
`Heterosis
`per sea
`
`%
`heterosisa
`
`4.1
`198.0
`
`117.0
`37.8
`0.5
`0.3
`11.7
`16.3
`2.1
`
`3.0
`0.4
`6.9
`
`8.6
`
`0.3
`
`64.7%
`46.0%
`
`101.7%
`21.8%
`3.4%
`3.4%
`15.5%
`18.1%
`2.9%
`
`4.3%
`4.8%
`48.3%
`
`26.5%
`
`69.0%
`
`aBetter-parent heterosis.
`bZanoni and Dudley (1989).
`cUnpublished data, N.M. Springer and R.S. Stupar.
`dAuger et al. (2005a).
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`Figure 1. Phenotypic heterosis in the B73 ⳯ Mo17 hybrid. Represen-
`tative B73, Mo17, and F1 hybrid ears and plants are shown. Note the
`increased size of the two hybrid ears and three hybrid rows relative to the
`two inbred parents (left, Mo17; right, B73).
`
`this review focuses on the heterosis derived from crossing maize
`inbred line B73 with inbred line Mo17 (Fig. 1). The B73 ⳯ Mo17
`hybrid was widely grown commercially in the United States dur-
`ing the 1970s, with >15 million bags of seed sold, sufficient to
`plant ∼45 million acres (Troyer 2006). While there are many
`maize hybrids that exhibit high levels of heterosis, this particular
`hybrid provides an excellent example of heterosis involving two
`lines that are relatively well-characterized in terms of genetic
`mapping, gene expression, and genome organization.
`The relative measurements for several traits of the B73 and
`Mo17 parental inbred lines and the hybrid are listed in Table 1
`(Zanoni and Dudley 1989; Auger et al. 2005b). Note that the
`quantitative measurements of heterosis vary significantly for dif-
`ferent traits. The variation in levels of heterosis among traits
`leads to difficulties in accurately quantifying the amount of over-
`all heterosis. There is also variation in the relative level of het-
`erosis for different traits between different hybrids. For example,
`one maize hybrid may exhibit significant heterosis for plant
`height but have low heterosis for grain yield, while a second
`hybrid may exhibit high heterosis for grain yield but very low
`heterosis for height (Zanoni and Dudley 1989; data not shown).
`This variation suggests that the same set of genes does not con-
`trol all heterotic responses. Additionally, heterosis does not sim-
`ply result from the overall genetic diversity within a hybrid, but
`is likely a reflection of diversity at specific, important genes that
`contribute to a particular trait. This view is supported by the
`ability to map QTLs that contribute to heterosis for individual
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`Text Box 1. Dominance and overdominance models of
`heterosis
`
`The genetic basis of heterosis has been debated for nearly a cen-
`tury without a clear resolution. The two main hypotheses ad-
`vanced to explain this phenomenon are dominance (Davenport
`1908; Jones 1917) and overdominance (East 1908; Shull 1908).
`These models have survived with various modifications and inter-
`pretations as the methods and specialties of biology have
`changed (Crow 1999; Birchler et al. 2003). The dominance model
`of heterosis posits that each of the inbred lines contains slightly
`deleterious alleles that reduce their fitness. The hybrid will benefit
`from the complementation of these deleterious alleles and will
`display a superior phenotype. The overdominance model sug-
`gests that the heterozygous combination of alleles at a given locus
`is phenotypically superior to either of the homozygous combina-
`tions for that locus; contributions of overdominant loci thereby
`result in a superior hybrid.
`Genetic linkage causes major difficulties in resolving dominant
`from overdominant action on phenotypes. A locus may display
`apparent overdominant action that is actually the result of
`pseudo-overdominance, which occurs when two repulsion-phase-
`linked loci both contribute via dominant mechanisms (Stuber et
`al. 1992; Graham et al. 1997). Bingham (1998) provided a dis-
`cussion of the role of chromosomal blocks and the ability to re-
`solve overdominance from pseudo-overdominance. In maize, it is
`likely that only 30 chromosomal blocks (a segment of a chromo-
`some derived from one parent) are transmitted (∼2 or 3 crossovers
`per chromosome pair). Given this limited resolution of recombi-
`nation at the individual level, it is anticipated that many repulsion-
`phase linkages will not be broken, resulting in pseudo-
`overdominance. The use of advanced populations in maize de-
`rived from random intermating provided strong evidence to
`support widespread pseudo-overdominance, as the evidence for
`overdominant loci was reduced over successive generations (for
`reviews, see Bingham 1998; Crow 1999).
`Although the current view favors dominant allelic action as the
`major contributor to heterosis (Coors and Pandey 1999; Crow
`1999), there are several lines of evidence that suggest that mecha-
`nisms beyond simple complementation may be important in het-
`erosis. The absence of a decline in the magnitude of heterosis
`(Duvick 2001), the progressive heterosis in tetraploids, and the
`rapid rate of inbreeding depression in tetraploids have been cited
`as factors that suggest that dominance may be insufficient to
`completely explain heterosis (Birchler et al. 2003). In addition, the
`existence of a locus with overdominant phenotypic action has
`been demonstrated in maize (Hollick and Chandler 1998). A re-
`cent study using introgression lines documented the contribution
`of dominance and overdominance to heterosis in tomato (Semel
`et al. 2006). This study found significant contributions of over-
`dominant action to reproductive traits but not other traits. How-
`ever, reproductive traits were also influenced by a larger number
`of QTL than other traits, which suggests a potential contribution
`of pseudo-overdominance.
`The dominance/overdominance debate becomes even more
`nuanced when contributions of epistasis are considered. Epistasis
`is classically defined as interactions between genes at two (or
`more) loci affecting the phenotypic expression of a trait. With
`regard to heterosis, a dominant-acting locus may interact with an
`overdominant-acting locus to further heterotic gain. Therefore,
`the genetic background and allelic interactions therein can have
`an effect on the heterotic contributions of individual loci.
`
`absence of variation (inbreds), there is no heterosis. Our assump-
`tions of the nature and frequency of allelic variation can limit
`how we consider heterosis (Crow 1999; Phillips 1999).
`
`Allelic diversity in maize genic sequences
`
`One of the most common approaches toward documenting alle-
`lic diversity is to compare the sequence of genic regions (includ-
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`266 Genome Research
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`ing coding regions, introns, untranslated regions, and single-
`copy DNA surrounding genes) from multiple strains or varieties
`in order to identify variation. This variation can then be used for
`mapping or association studies. An investigation of randomly
`selected sequences in the maize inbred B73 relative to inbred
`Mo17 found that, on average, indel polymorphisms occur every
`309 bp and SNPs occur every 79 bp (Vroh Bi et al. 2005). The
`analysis of 300–500 bp amplicons found that 44% of the se-
`quences contained at least one polymorphism in B73 relative to
`Mo17. In general, it is estimated that there is one polymorphism
`every 100 bp in any two randomly chosen maize inbred lines
`(Tenaillon et al. 2001; Ching et al. 2002). Sequence diversity data
`have been evaluated for several hundred diverse inbred lines of
`maize at >3000 genic sites (Zhao et al. 2006). These data provide
`extensive information about the SNP and indel frequencies of
`maize alleles. Collectively, these studies indicate that maize has a
`relatively high level of sequence polymorphism compared to
`many other species. For example, the level of sequence diversity
`in genic sequences within maize is estimated to be higher than
`the level of diversity between humans and chimpanzees (Buckler
`et al. 2006).
`
`Structural diversity in maize
`
`Structural genome diversity involves alterations in DNA se-
`quence beyond SNPs or small indels. This would include large-
`scale chromosomal differences, altered location of genes or re-
`petitive elements, or differences in the presence of sequences in
`one inbred relative to another. Large-scale genome differences
`between different maize inbred lines were first identified through
`cytogenetics studies. Barbara McClintock and others analyzed
`heterochromatic knob (highly condensed, tandem repeat re-
`gions) content and size to characterize genome variation in
`maize (Brown 1949; McClintock et al. 1981; Adawy et al. 2004).
`Recent studies have documented differences in the content for
`several classes of repetitive DNA between maize inbreds at the
`chromosomal level (Kato et al. 2004). Flow cytometry studies
`have also documented significant variation in the size of the
`maize genome between different inbred lines (Laurie and Bennett
`1985; Lee et al. 2002).
`Sequence-based methodologies have documented maize ge-
`nome structural diversity at a higher resolution (for reviews, see
`Buckler et al. 2006; Messing and Dooner 2006). BAC-based stud-
`ies focusing on the diversity of allelic regions among different
`maize inbred lines identified a surprising level of intraspecific
`structural diversity at several loci (Fig. 2). Fu and Dooner (2002)
`sequenced BAC contigs that contain the bz gene from the inbred
`B73 and McC genotypes and, as expected, documented numer-
`ous SNP and short indel polymorphisms within the genes pres-
`ent in this region. In addition, they found a surprising level of
`structural diversity in the two regions that resulted in extensive
`sequence nonhomologies. This structural diversity includes
`variation in repetitive elements and variation in the presence of
`genic fragments.
`Seven conserved genes were found in the bz region of chro-
`mosome 9 for both B73 and McC; however, an additional four
`gene-like fragments were found only in the McC haplotype (Fu
`and Dooner 2002). Initially these fragments were thought to be
`genes specific to the McC genotype. However, further investiga-
`tion found that these fragments are actually partial gene seg-
`ments that were likely the result of two transposition events me-
`diated by Helitron transposable elements (Lai et al. 2005). The
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`Allelic variation and heterosis
`
`Figure 2. Potential examples of allelic variation in maize. A hypothetical small chromosomal region is diagrammed for B73 and Mo17. The genes (red)
`are separated by clusters of transposons (black) and several different classes of retrotransposons (various shades of green). There are likely to be
`numerous SNPs and small indels within this region. The composition of repetitive sequences also shows significant variation between the two inbreds.
`The specific location of a causative change that has resulted in functional allelic variation is indicated by the yellow lightening bolts. Gene A is an example
`of a variant in which the proteins are different. Gene B is a nonshared sequence that is only present in B73 at this locus. Gene C is more highly expressed
`in Mo17 than in B73 because of differences in the repetitive elements surrounding this gene in B73 and Mo17. Gene D is expressed only in B73 and
`not in Mo17 because of altered sequence in nearby regulatory regions. Gene E shows no functional variation in B73 relative to Mo17.
`
`partial fragments present at the McC bz locus on chromosome 9
`are derived from full-length gene sequences that are present on
`chromosome 5 in both B73 and McC (Lai et al. 2005). Helitron
`transposons, which use a rolling-circle method of replication,
`exhibit limited primary sequence signatures and can be difficult
`to identify using computational approaches (Kapitonov and
`Jurka 2001). The exact mechanism through which these elements
`can “capture” and transpose genic fragments is not well-
`understood (Brunner et al. 2005b; Lai et al. 2005).
`The B73 and McC genotypes also demonstrated structural
`variation for repetitive elements present in this region (Fu and
`Dooner 2002). Only one short fragment from a repetitive Zeon1
`element is conserved in both genotypes. Otherwise, the trans-
`posable elements present in McC and B73 are completely distinct
`in terms of their relative positions. The McC haplotype contains
`six full-length LTR retrotransposons and six partial elements that
`are not conserved in the B73 locus, while the B73 haplotype
`contains five full-length LTR retrotransposons and four partial
`elements that are not present in the McC locus (Fu and Dooner
`2002). In many cases, the B73 and McC alleles of the same gene
`were surrounded by distinct repetitive elements that may con-
`tribute to differences in the chromatin neighborhood for the two
`alleles. A recent follow-up study compared the genomic sequence
`structure of the bz locus in eight different maize lines represent-
`ing a wide range of geographic and genetic backgrounds (Wang
`and Dooner 2006). A tremendous degree of variation in genomic
`sequence structure was identified among these lines; the authors
`concluded that, on average, any two of the eight bz haplotypes
`share just 50% of their sequences (Wang and Dooner 2006).
`Several other maize loci that were sequenced in multiple
`inbred lines also exhibit structural diversity for genic fragments
`and repetitive elements. The tandemly repeated zein1C gene fam-
`ily displays variation between maize inbred lines in terms of copy
`number and expression (Song and Messing 2003). The sequenc-
`ing of four genomic loci in B73 and Mo17 identified 45 genes
`that were shared between genotypes, while another 27 genic frag-
`ments (which are likely pseudogenes) were only present in either
`one of the two inbred lines (Brunner et al. 2005a). These four loci
`also included a set of 27 LTR retroelements that are present at
`collinear locations in both B73 and Mo17, while another 62 LTR
`
`retroelements were only present in either one of the two inbred
`lines.
`The genome-wide prevalence of nonshared genic fragments
`was investigated using BAC library hybridization methods
`(Morgante et al. 2005). Hybridization of short genic probes to
`fingerprinted BAC libraries from B73 and Mo17 resulted in
`mapping ∼20,500 genes, of which 80% were present in both in-
`breds, 11% were specific to B73, and 9% were specific to Mo17.
`Applying these frequencies to the anticipated genome size of
`maize suggests that the B73 and Mo17 genomes may contain
`∼10,000 nonshared genic fragments; a subset of which may be
`functional.
`There is evidence that mechanisms other than Helitron-
`mediated transposition can lead to the presence of nonshared
`genic fragments in different varieties of a species (Bennetzen
`2005). For instance, other classes of transposons can modify
`genic content. Mutator-like transposons can “capture” genic se-
`quences and move these sequences within the maize genomes
`(Bureau et al. 1994; Jin and Bennetzen 1994; Bennetzen 2005).
`The abundance of the Mutator-like elements that carry gene frag-
`ments, termed Pack-MULEs, is not known in maize; however, the
`completion of the rice genome has allowed for a comprehensive
`analysis of these elements in rice (Jiang et al. 2004). Fragments of
`>1000 genes have been captured and sometimes duplicated by
`Pack-MULEs, resulting in the presence of >3000 Pack-MULEs in
`rice. There is EST evidence for the expression of ∼5% of the Pack-
`MULEs, and some of these expressed sequences may encode
`functional peptides, although most contain numerous stop
`codons. There is also evidence for the presence of large numbers
`of tandem duplications in the maize genome, including some
`examples of tandem duplications that are present only in some
`inbred lines (Emrich et al. 2007). Duplicated genes and trans-
`posed gene fragments, like those discussed above, are likely to
`show variation within a species such that specific duplications or
`Pack-MULEs will be present within one inbred and absent in
`others.
`The presence of nonshared genic sequences or retrotrans-
`posons might effect phenotypic variation through a variety of
`mechanisms. The majority of examples of the nonshared genic
`sequences that have been characterized to date are partial gene
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`fragments. It remains possible that some of these nonshared
`genic sequences will include full-length genes or novel genes
`created by shuffling of exons from multiple genes (Brunner et al.
`2005b). In fact, there is evidence for expression of some of these
`nonshared genes (Brunner et al. 2005b; Morgante et al. 2005).
`The expression level and pattern of these fragments are likely to
`be distinct from those of the original gene because the newly
`transposed gene copy often does not include the full promoter or
`regulatory sequences. In some cases, the transcription and trans-
`lation of a transposed partial ORF may result in an aberrant pro-
`tein that has a dominant-negative effect that inhibits the func-
`tion of the original full-length protein.
`The variation in maize repetitive elements may influence
`the expression level of nearby genes, and in some cases, this
`influence can extend over large regions. For example, most alleles
`of the maize transcription factor B have relatively small, closely
`linked, regulatory sequences. However, the transcription level of
`the B-I allele can be influenced in cis by a set of tandem repeats
`that are located >100 kb upstream of the coding region (Stam et
`al. 2002). Additionally, the tb1 locus, which influences maize
`architecture, is influenced in cis by a region of repetitive elements
`>50 kb upstream of the gene (Clark et al. 2006). These examples
`suggest that in some cases, repetitive sequences may affect the
`expression level of maize genes, ultimately resulting in altered
`phenotypes. However, it is unknown whether these examples
`represent rare cases of long-distance effects of repetitive se-
`quences, or if this phenomenon is common in maize gene regu-
`lation.
`
`Expression diversity in maize
`
`Intraspecific allelic variation also includes variation in gene ex-
`pression. Several recent studies have documented expression di-
`versity in maize. Analysis of gene expression levels using an Ag-
`ilent long oligonucleotide microarray platform found evidence
`for the differential expression of ∼5% of genes in the maize in-
`bred lines A619 and W23 at several developmental stages (Ma et
`al. 2006). A cDNA microarray platform used by Swanson-Wagner
`et al. (2006) identified ∼10% of genes as being differentially ex-
`pressed in B73 and Mo17 seedlings. Affymetrix microarray analy-
`sis also found prevalent differential expression between B73 and
`Mo17 in seedling, immature ear, and embryo tissues (Stupar and
`Springer 2006).
`The Affymetrix microarray study suggested that ∼2.5% of
`the genes present on the array were detected in only one of the
`two transcriptomes, either B73 or Mo17 (Stupar and Springer
`2006). Validation of these results provided evidence that many of
`these differences in B73–Mo17 transcriptome content are not the
`result of differences in genomic content. Instead, these presence–
`absence transcriptome differences appear to be the result of dif-
`ferentially expressed genes that are present in the genomes of
`both B73 and Mo17.
`The expression diversity observed in maize could be the re-
`sult of cis-acting variation at each of the differentially expressed
`genes or the result of variation at a small number of trans-acting
`loci that have downstream regulatory effects. Cis-acting variation
`can be the result of alterations in regulatory sequences (i.e., en-
`hancers and promoters), sequence changes that affect the RNA
`stability, or heritable variation in chromatin structure. Trans-
`acting variation can be the result of quantitative or qualitative
`variation in a factor that influences the expression of the gene,
`such as a transcription factor. The prevalence of cis- and trans-
`
`268 Genome Research
`www.genome.org
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`regulatory variation can be assessed using expression quantita-
`tive trait loci (eQTL) (Cheung and Spielman, 2002) and allele-
`specific expression (ASE) (Wittkopp et al. 2004) analyses. Numer-
`ous studies in animal systems using both eQTL (Monks et al.
`2004; Morley et al. 2004; Cheung et al. 2005; Doss et al. 2005;
`Pastinen et al. 2005; Stranger et al. 2005) and ASE (Cowles et al.
`2002; Yan et al. 2002; Bray et al. 2003; Lo et al. 2003; Pastinen et
`al. 2004; Wittkopp et al. 2004) have found that a significant
`amount of expression diversity is the result of cis-acting variation.
`There is evidence for prevalent cis-acting regulatory varia-
`tion in maize. An eQTL study found that 80% of the eQTL with
`a LOD score >7.0 mapped to the same physical location as the
`differentially expressed gene, indicating that allelic cis-variation
`may be causing regulatory differences for many maize genes
`(Schadt et al. 2003). Guo et al. (2004) used ASE to study the
`relative expression of two alleles in maize F1 hybrids. Both alleles
`present in an F1 hybrid have access to an identical set of trans-
`acting factors, thus unequal, or biased, allelic expression suggests
`cis-acting variation between alleles. Frequent allelic expression
`bias in F1 hybrids was observed (11/15 genes), suggesting that
`cis-variation is present in many maize genes (Guo et al. 2004).
`These findings were supported by another ASE study that found
`allelic cis-variation contributing to inbred expression differences
`in 46 of 53 maize genes (Stupar and Springer 2006).
`In addition to structural and expression diversity, there are
`additional sources of diversity within maize. Variation in sense–
`antisense transcription, allelic variation in DNA methylation pat-
`terns, and allelic variation in chromatin structure are all topics
`that have not been extensively addressed to date. One study of
`the targets of CpNpG methylation in maize found substantial
`variation for the epialleles present in B73 and Mo17 (I. Makare-
`vitch and N.M. Springer, unpubl.). In addition, studies of poly-
`ploids have found evidence for widespread epigenetic alterations
`that can lead to phenotypic variation (Osborn et al. 2003). The
`epigenetic diversity within a species may be particularly impor-
`tant in contributing to overdominance. For example, one of the
`few examples documenting overdominant gene action for a
`single locus involves an epiallele at the Pl locus in maize (Hollick
`and Chandler, 1998). Phillips (1999) presented an excellent dis-
`cussion of the evidence for de novo variation in inbred lines and
`how this variation may contribute to heterosis.
`
`Interactions of alleles in hybrids
`
`The allelic variation described above is brought together in the
`hybrid organism. The allelic combinations present in a hybrid
`may result in interactions that alter expression profiles, new pro-
`tein–protein interactions, or epistatic interactions. The studies of
`gene expression in inbreds and hybrids can be divided according
`to whether they have found substantial evidence for frequent
`nonadditive expression patterns compared to the expression lev-
`els in the inbred parents (see Text Box 2 for a primer on the
`terminology and concepts associated with gene expression in
`inbreds relative to hybrids). The concept of transcriptional epis-
`tasis and the evidence for this phenomenon in hybrid maize are
`also discussed.
`
`Evidence for allelic interactions that lead to