Skip to main content
NCBI home page
Genetics logoLink to Genetics
. 2005 Nov;171(3):1289–1303. doi: 10.1534/genetics.105.044552

Genetic Mapping of Species Boundaries in Louisiana Irises Using IRRE Retrotransposon Display Markers

Amy Bouck *,1, Ryan Peeler †, Michael L Arnold *, Susan R Wessler †
PMCID: PMC1456832  PMID: 16079236

Abstract

Genetic mapping studies provide insight into the pattern and extent of genetic incompatibilities affecting hybridization between closely related species. Genetic maps of two species of Louisiana Irises, Iris fulva and I. brevicaulis, were constructed from transposon-based molecular markers segregating in reciprocal backcross (BC1) interspecific hybrids and used to investigate genomic patterns of species barriers inhibiting introgression. Linkage mapping analyses indicated very little genetic incompatibility between I. fulva and I. brevicaulis in the form of map regions exhibiting transmission ratio distortion, and this was confirmed using a Bayesian multipoint mapping analysis. These results demonstrate the utility of transposon-based marker systems for genetic mapping studies of wild plant species and indicate that the genomes of I. fulva and I. brevicaulis are highly permeable to gene flow and introgression from one another via backcrossing.

NATURAL hybridization is the interbreeding of genetically differentiated populations or species in the wild (Arnold 1997 as adapted from Harrison 1990), and it has been increasingly recognized as an important force in evolution (Arnold 1997; Rieseberg and Carney 1998; Rieseberg et al. 2003; Seehausen 2004). Hybridization contributes to genetic variation and evolution in numerous ways. It can serve as an avenue for the transfer of additive genetic variation that may contribute to adaptation, a process known as adaptive introgression (Anderson 1949; Arnold 1997; Grant and Grant 2002). Hybrid lineages may also form stable species that are distinct and reproductively isolated from the original parent taxa (Rieseberg et al. 2003). If hybrid progeny suffer from reduced fitness, selection may lead to reinforcement of reproductive isolation (Dobzhansky 1940; Blair 1955; Howard 1993; Marshall et al. 2002). Alternatively, hybridization may lead to the loss of genetic and phenotypic differentiation as one species is assimilated into another (Rhymer and Simberloff 1996; Huxel 1999; Anttila et al. 2000).

The evolutionary outcome of hybridization is in part determined by the nature and extent of species barriers, particularly genetic interactions that may occur between divergent genomes. Hybridization combines alleles that did not evolve in concert. A well-developed body of theory (Bateson 1909; Dobzhansky 1936, 1937; Muller 1940; Orr 1995; Turelli and Orr 2000; Orr and Turelli 2001) and experimental work (Fishman and Willis 2001; Harushima et al. 2001, 2002; Presgraves 2003; Presgraves et al. 2003; Barbash et al. 2004) has shown how this can result in genetic incompatibilities due to negative epistatic interactions between divergent alleles at different loci, resulting in hybrid genotypes with reduced fitness. Alleles or chromosomal regions contributing to reduced hybrid fitness will be less likely to introgress across species boundaries than those with neutral or positive fitness effects (Key 1968; Barton and Hewitt 1985; Navarro and Barton 2003). This means that reproductive isolation varies across the genome and may be very different from chromosome to chromosome, locus to locus, or even allele to allele (Key 1968; Barton and Hewitt 1985; Harrison 1990; Buerkle and Rieseberg 2001). As a result, the pattern and extent of genetic interactions that occur in hybrid genotypes sets the stage for whatever evolutionary outcomes ensue.

The Louisiana Irises (Iridaceae, section Apogon, series Hexagonae) have been the focus of numerous studies of interspecific hybridization and adaptive introgression (Small and Alexander 1931; Viosca 1935; Anderson 1948; Arnold et al. 2001; Johnston et al. 2003). These are long-lived, self-compatible, rhizomatous, perennial herbs that reproduce both sexually and clonally. Two members of this clade, Iris fulva and I. brevicaulis, are broadly sympatric throughout the Mississippi River valley of central North America, but are found in different habitats (Viosca 1935; Cruzan and Arnold 1993; Johnston et al. 2001b). I. fulva is found in intermittently flooded, forested wetlands throughout this range, while I. brevicaulis is normally found in drier, riparian-associated hardwood forests. I. brevicaulis is primarily bumble bee pollinated and possesses a suite of classic bee-pollination-associated floral traits: blue flowers marked with prominent white and yellow nectar guides, stiff upright tepals, and strongly scented flowers (Viosca 1935; Wesselingh and Arnold 2000). I. fulva is primarily hummingbird pollinated and possesses a markedly different floral phenotype: flowers of this species are solid red with protruding anthers and highly reflexed tepals (Viosca 1935; Wesselingh and Arnold 2000). I. fulva and I. brevicaulis also differ in flowering time: I. fulva populations begin flowering about a month earlier than I. brevicaulis populations in southern Louisiana (Cruzan and Arnold 1994).

Despite these differences in habitat, floral morphology, and phenology, these species form numerous hybrid zones in southern Louisiana, often as a consequence of habitat disturbance (Viosca 1935; Anderson 1948). Previous studies have detected extensive introgression of genetic markers in natural hybrid populations (Arnold et al. 1992; Cruzan and Arnold 1993; Johnston et al. 2001b). Still, these species remain morphologically distinct: genetic analyses of hybrid zones have indicated that the species mate assortively and that embryos with intermediate hybrid genotypes are more likely to be aborted (Cruzan and Arnold 1994). Consistent with a pattern of genetic incompatibilities in some hybrid genotypes is the occurrence of wide variation in pollen fertility of wild hybrid plants (Randolph et al. 1967). In addition, multilocus genetic incompatibilities have been detected in experimental F2 hybrids (Burke et al. 1998). Initial formation of F1 hybrids between I. fulva and I. brevicaulis appears to be extremely rare, and hybrid zone populations typically consist of genotypes resembling advanced generation backcross hybrids. This is in spite of the fact that F1 hybrids of I. fulva and I. brevicaulis are vigorous and highly fertile (Johnston et al. 2003) and undergo normal meiosis. Pollinator studies have shown that gene flow via pollinator movements between F1's and parental species types is likely to be high (Wesselingh and Arnold 2000).

In summary, previous results have detected genetic incompatibilities between I. fulva and I. brevicaulis, but the widespread occurrence of advanced generation hybrid plants in wild populations indicates that some genomic regions of these species must be permeable to introgression. This article presents a study assessing genetic interactions affecting hybridization between I. fulva and I. brevicaulis in reciprocal backcross 1 (BC1) hybrids, which are a realistic model for hybrid genotypes likely to form in natural hybrid populations.

Studies of genetic incompatibilities between crop species and their relatives have taken advantage of well-saturated genetic maps available in agriculturally or economically important plants. These studies have provided a detailed picture of the genomic pattern of species barriers that limit introgression among cultivated subspecies [e.g., rice (Harushima et al. 2001, 2002) and cotton (Jiang et al. 2000)], crops and their wild relatives [e.g., tomato (Bernacchi and Tanksley 1997; Moyle and Graham 2005) and corn (Doebley et al. 1995)], and congeners with economically desirable traits [e.g., eucalyptus (Myburg et al. 2004)]. In contrast, there are few studies of introgression in wild plant species at the level of detail provided by a genetic mapping approach. A number of these studies have focused on QTL mapping of the genetic basis of prezygotic barriers to gene flow in Mimulus (e.g., Bradshaw et al. 1995, 1998; Lin and Ritland 1997; Fishman et al. 2002). Genetic mapping studies also provide a means of assessing the number and distribution of genetic factors that affect hybridization, including the identification of genomic regions permeable or resistant to introgression, or that directly contribute to breakdown in fitness components [e.g., between species of Helianthus (Rieseberg et al. 1995, 1999; Kim and Rieseberg 1999; Buerkle and Rieseberg 2001)].

Genetic mapping studies require large numbers of molecular markers. Louisiana irises have a large physical genome with a haploid genome size of ∼10,000 Mb (Kentner et al. 2003). In species with large genomes, the predominance of repetitive DNA may hamper the use of standard amplified fragment length polymorphisms (AFLPs) or RFLP- and microsatellite-based markers (Kinlaw and Neale 1997; Pfeiffer et al. 1997; Remington et al. 1999). Transposable element markers based on IRRE retrotransposons were developed for use in Iris by Kentner et al. (2003). The IRRE long terminal repeat (LTR) retrotransposons are a group of TY3/gypsy-like elements that compose 6–10% of the Louisiana Iris genome (Kentner et al. 2003). A modification of the amplified fragment length polymorphisms (Vos et al. 1995) procedure called transposon display is used to generate PCR products that are anchored in a transposable element and a flanking restriction site (Waugh et al. 1997; Ellis et al. 1998; Van den Broeck et al. 1998; Casa et al. 2000). The utility of LTR retrotransposon display markers has been demonstrated previously by mapping studies of, for example, barley (Waugh et al. 1997; Manninen et al. 2000) and pea (Ellis et al. 1998; Flavell et al. 1998; Kalendar et al. 1999). This article presents genetic maps of I. fulva and I. brevicaulis constructed from IRRE retrotransposon markers segregating in interspecific BC1 hybrids. To identify genomic regions in which introgression of heterospecific alleles may be reduced or promoted, patterns of transmission ratio distortion (TRD) at marker loci were examined. The transmission ratios of parental alleles at marker loci were compared to expected Mendelian segregation and analyzed using the Bayesian approach of Vogl and Xu (2000). Mapping and segregation analyses indicate that the majority of the genomes of both I. fulva and I. brevicaulis appear highly permeable to introgression from one another via backcrossing.

MATERIALS AND METHODS

Generation of BC1 mapping populations:

One wild-collected individual from each of the two species was used to make reciprocal interspecific backcross 1 (BC1) mapping populations. In these crosses, the same individuals were used as both the F1 parents and the recurrent parents for backcrossing. This was done to minimize the within-species genetic variation segregating in the experiment. The individuals used were collected from natural populations in southern Louisiana that had apparently not experienced hybridization with the other species, as determined by population genetic analysis or by population observations. The I. fulva individual, If174, was collected from a population in Terrebonne Parish, and the I. brevicaulis individual, Ib72, was collected from a population in St. Martin Parish. Flowers of Ib72 were crossed with pollen from If174 to make F1 hybrids in 1997. Two F1 hybrids, designated F1(2) and F1(3), were used to make backcross hybrids during the winter of 1999. Pollen from F1(3) was crossed onto flowers of several ramets (clones) of Ib72 to make I. brevicaulis BC1 hybrids (IbBC1), and pollen from F1(2) was crossed onto flowers of several ramets of If174 to make I. fulva BC1 hybrids (IfBC1). All flowers were emasculated prior to opening, and pollen was applied 2 days later after the stigmatic surface became receptive. All plants were housed in the University of Georgia Department of Plant Biology greenhouses.

Seeds were planted in flats during the following summer and monitored for germination for ∼12 months. Seedlings were transplanted into 6-in. azalea pots shortly after germination. During September of 2000, a single rhizome of each BC1 genotype was transplanted in an 8-in. azalea pot. All BC1 plants as well as replicates of each parental genotype were randomly assigned positions in a single greenhouse. Plants were repotted annually by cutting out a single rhizome of each genotype and transplanting it to a new pot. Plants were randomly reassigned new positions each year at repotting. Mapping populations consisted of 230 IbBC1 and ∼225 IfBC1 hybrid plants. All of the IbBC1 plants and a subset of 120 of the IfBC1 plants were used in the present study.

Genetic mapping:

DNA isolation:

Leaf tissue samples were collected from plants during the fall of 200l at which point plants had been cultivated in the greenhouse for ∼18 months. Tissue samples were immediately snap frozen in liquid nitrogen. DNA was isolated using a modified CTAB protocol (Doyle and Doyle 1987; Soltis et al. 1991). DNA samples were treated with RNase A and standardized by comparison with λDNA of known concentration visualized on a 1% agarose gel with ethidium bromide.

Generation of transposon display genetic markers:

Genetic markers were generated using the IRRE transposon display marker protocol of Kentner et al. (2003). This marker system employs a modified AFLP protocol to assay polymorphism in DNA sequence flanking insertion sites of IRRE LTR retrotransposons.

Genomic DNA (∼500 ng) was digested with 50 units of EcoRI (New England Biolabs, Beverly, MA) at 37° for ∼12 hr. Standard EcoRI AFLP adapters (Vos et al. 1995) were ligated to the digested DNA for ∼12 hr at room temperature using 5 units of T4 DNA ligase and the manufacturer-supplied buffer (Invitrogen, San Diego).

Two rounds of PCR were performed: the first (preamplification) used a primer complementary to the EcoRI adapter sequence plus two selective bases, along with a primer specific to the 5′-end of the LTR of the IRRE1-A1 (CCA AAC CAA ACC AAG CCA CAC TAA ACC) or IRRE1-C (ACA GGA ACA CTT TCC AAT TAC GT) retrotransposon (Kentner et al. 2003). Preamplification PCR was performed in 30-μl reaction volumes using 1 μl of restriction/ligation reaction product, 10 pmol of each primer, 1.5 units of AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA), 0.2 μm of each dNTP, 3.0 μm MgCl2, and the manufacturer's buffer. PCR conditions were as follows: 72° for 2 min, 94° for 3 min, 35 cycles of 94° for 30 sec, 56° (IRRE1-A1) or 57° (IRRE1-C) for 30 sec, and 72° for 1 min, with a final elongation step at 72° for 3 min.

The second (selective) round of PCR amplification used EcoRI adapter primers plus four selective bases and IRRE1-A1 or IRRE1-C LTR-specific primers nested within those used in the preamplification reactions (Kentner et al. 2003). Selective PCR reactions were performed in 10-μl volumes using 1.5 μl of 10:1 diluted preamplification product as template, 5 pmol of EcoRI adapter, 3 pmol 33P-labeled IRRE1-A1 (CGT ATA AAA TAC GTA CAC AAG AG) or IRRE1-C (TCC AAT TAC GTA TAA AAT ACG) primer, 1.5 units of AmpliTaq DNA polymerase (Applied Biosystems), 0.2 μm of each dNTP, 2.5 μm MgCl2, and the buffer supplied with the enzyme. The cycling conditions were as follows: 94° for 3 min, 35 cycles of 94° for 30 sec, 56° for 50 sec, and 72° for 1 min, with a final elongation step at 72° for 4 min. PCR products were resolved on a 6% polyacrylamide sequencing gel and visualized by autoradiography. Bands were scored by eye for genetic analysis.

Estimation of heterozygosity at IRRE transposon display loci:

Heterozygosity (He) of the Ib72 and If174 parents was assessed directly by scoring segregation of all alleles in recurrent backcross progeny. In other words, heterozygosity of the Ib72 parent allele markers was estimated by observing the segregation of these alleles in the IbBC1's, for which Ib72 served as the recurrent parent. Likewise, the heterozygosity of If174 was determined by observing the segregation of If174 alleles in IfBC1 hybrids. This method does underestimate the heterozygosity of the recurrent parent because bands heterozygous in the recurrent parent but homozygous present in the F1 parent will be scored as homozygous present in the BC1 progeny. All bands that were clearly present in the parent lanes were scored to estimate heterozygosity whether or not they were subsequently used in linkage mapping and whether or not they were species specific (Figure 1). Heterozygosity was calculated as the proportion of loci for which segregation was observed.

Figure 1.

Figure 1.

Open in a new tab

Example of IRRE transposon display markers segregating in IbBC1 hybrids. Replicate samples of the introgressed If174 parent are in lanes 5 and 7, replicate samples of the F1(3) parent are in lanes 9 and 11, and replicate samples of the recurrent Ib72 parent are in lanes 13 and 15. F1(2) is in lanes 1 and 3; remaining lanes are IbBC1 hybrids. Examples of segregating If174 allele markers used in linkage mapping of I. fulva are indicated by arrows in lane 5. Examples of Ib72 alleles scored for estimating observed heterozygosity are indicated by arrows in lane 15. Size markers to the left are in base pairs.

Genetic map construction:

Linkage maps were constructed with Mapmaker 3.0 (Lander et al. 1987; Lincoln et al. 1992) by completing several rounds of grouping and marker exclusion. For all calculations involving map distances we used the Kosambi mapping function. Marker grouping was initiated using the GROUP command with a LOD of 11.0 and a maximum mapping distance of 37 cM. The LOD threshold was then dropped in a stepwise fashion down to a LOD of 6.0, resulting in 22 putative linkage groups incorporating 331 markers in the I. fulva (IbBC1) mapping population and 22 putative linkage groups incorporating 206 markers for the I. brevicaulis mapping population (IfBC1's). For each putative linkage group, all pairwise recombination fractions were scrutinized to ensure that no groups had been erroneously linked and to search for markers with conflicting linkage. In most cases a single anchor marker was designated for each linkage group using the ANCHOR command: in the case of the largest linkage groups, two anchor markers were used. Remaining loci were assigned to linkage groups using the ASSIGN command at a LOD of 5.0 and a maximum mapping distance of 37.0 cM. Linkage of any loci remaining unassigned was examined at decreasing LOD thresholds, holding the maximum mapping distance constant at 37.0 cM. Markers that could be unambiguously assigned to groups at lower LOD thresholds were kept in the data set; those showing conflicting linkage were discarded.

The marker order for each linkage group was established using the ORDER command of MAPMAKER with error detection on. The ORDER command was set to search for an initial order of four markers in most cases (three for smaller groups, five for larger groups) with a support-for-order LOD threshold of 3.0. Remaining markers were added to this initial order first at a LOD of 3.0 and then at a LOD of 2.0. Resulting marker orders were then examined using the RIPPLE command. In a few cases, marker orders were established with lower thresholds. Candidate errors identified by the ERROR DETECTION capability of the ORDER function were rescored, and loci with a high number of errors that could not be resolved were excluded from the framework map or discarded. The ordering procedure described above was then repeated after correcting genotyping errors and excluding high-error markers from the framework map. After this second round of ordering, the framework marker order for each linkage group was set using the FRAMEWORK command, and remaining markers were placed in their most likely interval using the PLACE function.

Marker spacing, genome length, and coverage estimation:

The average framework marker spacing, s, was calculated by dividing the summed length of all linkage groups by the number of intermarker intervals (equal to the number of framework markers minus the number of linkage groups) (Fishman et al. 2001). The total genome length, L, was estimated using two different methods to account for chromosome ends beyond the terminal markers of each linkage group. The first method added 2s to the length of all linkage groups. The second method, method 4 of Chakravarti et al. (1991), was employed by multiplying the length of each linkage group by the factor (m + 1)/(m − 1), where m is the number of framework markers on each group. The map coverage, c, was estimated as the percentage of the genome within d cM of a framework marker, where c = 1 − e (−2dn/L), and n is the number of framework markers (Chakravarti et al. 1991).

Following the method employed by Cervera et al. (2001), a coefficient of dispersion was calculated (Sokal and Rohlf 1995) to address whether IRRE display markers appear clustered in the linkage map. If the occurrence of an IRRE insertion site and marker locus is assumed to be a rare, independent event, then the frequency distribution of the number of marker loci occurring in each 10-cM interval should follow a Poisson distribution. To test this, the linkage map was divided into 10-cM intervals, and the number of intervals with 0–15 markers was counted. The coefficient of dispersion was calculated as CD = s2/Y, where Y is the mean number of markers per 10-cM interval, and s2 is the sample variance. A value of CD >1.0 indicates that loci are more clumped than would be expected if the occurrence of IRRE display loci are random and independent; a value <1.0 indicates that loci are more dispersed than would be expected.

Comparison to expected segregation ratios:

Agreement with expected Mendelian segregation ratios was evaluated using the χ2 goodness-of-fit test with the degrees of freedom equal to the number of observed genotypic classes (e.g., 2.0) minus 1.0 (Sokal and Rohlf 1995). Observed genotype frequencies of all marker loci incorporated into the linkage map were compared to expected genotype frequencies segregating in a 1:1 ratio. Each locus was tested individually, and the presence of distorted map regions was evaluated on the basis of the map distribution of loci showing significant TRD. Bonferroni corrections accounting for multiple tests were not used because genotype frequencies at individual marker loci may be related by linkage and therefore should not be expected to vary independently. Our choice to not correct for multiple tests may lead us to erroneously reject the null hypothesis at some loci, due to the fact that a small number of our numerous tests would be expected to surpass our significance threshold due to chance alone. However, our approach is more likely to allow us to detect transmission ratio distortion in map regions where distortion might actually exist but is not extreme or where there is low marker coverage.

Detection of map regions showing transmission ratio distortion:

To examine the pattern of TRD across the linkage map, we identified regions of two or more contiguous framework markers showing distortion in the same direction at α < 0.05 and α < 0.01. An indicator of the direction and magnitude of transmission ratio distortion was calculated as the relative frequency of the introgressed alleles: frequencies greater than or less than the expected value of 0.5 indicate over- or underrepresentation of the introgressed parent allele, respectively.

Bayesian mapping of transmission ratio distorting loci:

The Bayesian multipoint mapping method of Vogl and Xu (2000) was implemented as an additional means of detecting and localizing map regions of transmission ratio distortion. This method treats the number, location, and effects of transmission ratio distorting loci (TRDLs) as unknowns and makes use of reversible jump Markov chain Monte Carlo (MCMC) to estimate these variables. For the MCMC procedure, a Poisson prior of zero and a maximum of four (or, in the case of small linkage groups, two) TRDLs per linkage group was used. The chain length was set to 3000 iterations, which was determined to be sufficient by performing trial runs using various chain lengths (data not shown). The resulting number of TRDLs, their positions, and effects were treated as samples from the joint posterior distribution. Each linkage group was analyzed separately, and only linkage groups containing at least three markers were analyzed.

RESULTS

Generation of transposon display genetic markers:

A total of 48 IRRE transposon display primer combinations were tried in the IbBC1 mapping population, 38 of which were also run in the IfBC1 population (Table 1). Reactions based on the EcoRI + AG preselective primers were dropped because these reactions yielded fewer bands that were significantly less sharp than reactions based on EcoRI + AC or + CT primers. Transposon display reactions yield from 3 to 15 segregating If174-specific alleles (If alleles) and from 0 to 15 segregating Ib72-specific alleles (Ib alleles) (Figure 1). IRRE1-C- and IRRE1-A1-based reactions yield an average of 8.45 (N = 20) and 8.75 (N = 28) If alleles and 6.2 (N = 18) and 10.2 (N = 20) Ib alleles, respectively. The greater range in the number of Ib allele markers per reaction may be due in part to the higher heterozygosity (He) of the Ib72 parent. Overall heterozygosity, assessed for each parent by scoring segregation in progeny for which that genotype served as a recurrent parent, was 0.59 for Ib72 and 0.31 for If174.

TABLE 1.

Segregating IRRE transposon display polymorphisms by primer combination

EcoRI primer selective extension
IRRE-A1 element primer
IRRE-C element primer
I. fulva markers I. brevicaulis markers I. fulva markers I. brevicaulis markers
CTTT 6 14 5 0
CTTG 5 11 7 7
CTTC 7 15 8 8
CTTA 5 9 11 10
CTGT 15 14 7 10
CTGA 6 11 6 5
CTCT 7 10 8 7
CTCA 10 8 10 9
CTAT 13 10 8 11
CTAG 6 11 8 6
CTAC 12 12 13 14
CTAA 9 12 14 8
AGTT 6 — — —
AGTG 3 — — —
AGTC 8 — — —
AGTA 10 — — —
AGCA 8 — — —
AGAT 5 — — —
AGAC 8 — — —
AGAA 7 — — —
ACTT 13 4 7 1
ACTC 8 11 8 0
ACTA 14 9 8 2
ACGT 10 7 6 1
ACGA 14 9 8 —
ACAT 9 12 7 6
ACAC 9 6 12 1
ACAA 12 6 8 —
Total 245 201 169 106
Open in a new tab

Construction of the I. fulva linkage map using IbBC1 hybrids:

Linkage mapping of I. fulva using IbBC1 hybrids was based on a data set initially consisting of 414 IRRE transposon display markers genotyped in a subset of 120 progeny. Preliminary linkage analysis identified a group of 148 framework markers, which were then genotyped in the remaining 110 progeny subsequent to the final round of linkage map construction. A total of 401 markers were incorporated into the final I. fulva linkage map (Figure 2). The majority (344) of the 414 markers in the initial data set were assigned to linkage groups at LOD = 5.0 at a maximum mapping distance of 37.0 cM. Linkage grouping of the remaining 70 loci was completed at lower LOD thresholds, holding the maximum mapping distance constant at 37.0 cM. Seventeen loci were assigned to linkage groups at a LOD of 4.0, 14 were assigned at a LOD of 3.0, 18 assigned at a LOD of 2.0, and 8 at a LOD of 1.5. Finally, 6 markers were discarded due to conflicting linkage, 6 markers demonstrated linkage to groups at LODs <1.5 but were discarded, and 1 marker was completely unlinked.

Figure 2.

Figure 2.

Open in a new tab

Linkage map of I. fulva IRRE retrotransposon display markers segregating in IbBC1 hybrids. The first letter of each marker name (a or c) indicates IRRE-A1 and IRRE-C marker loci, respectively. The last four letters in each marker name indicate the EcoRI primer combination used to resolve the locus. Framework marker placements are indicated by a horizontal slash. Placed markers are shown in their most likely interval of location and are indicated by italics. Intermarker recombination distances are shown in Kosambi cemtimorgans. Markers showing statistically significant transmission ratio distortion are designated by x (α < 0.05) or a solid circle (α < 0.01).

The I. fulva linkage map consists of 142 framework markers on 22 linkage groups (Figure 2). Framework markers were those that could be ordered at a multipoint linkage analysis LOD threshold of at least 2.0. A total of 259 additional markers have been assigned to linkage groups and placed on the map but could not be assigned to a single interval at this statistical threshold.

The number of linkage groups resolved (22) agrees well with the cytologically determined haploid chromosome number of 21 (Randolph et al. 1961). The number of framework markers per linkage group ranges from 2 to 14, and linkage groups vary in length from 12 to 148 cM (Figure 2). The total length of all linkage groups resolved in the I. fulva linkage map is 1439 cM. The average marker spacing is 12 cM. Estimates of the total map length range from 1966 cM, calculated by adding 2s to each of the 22 linkage groups, to 1980 cM, calculated using method 4 of Chakravarti et al. (1991). This results in a coverage estimate of 76% of the genome within 10 cM of a framework marker. The coefficient of dispersion of 3.22 indicates that markers are clustered.

Construction of the I. brevicaulis linkage map using IfBC1 hybrids:

A total of 286 markers were incorporated into the I. brevicaulis linkage map (Figure 3). The majority (215) of the 307 markers in the initial data set were assigned at a LOD of 5.0 and a maximum mapping distance of 37.0 cM. Linkage of the remaining 92 loci was examined at decreasing LOD thresholds, holding the maximum mapping distance constant at 37.0 cM. Eleven loci were assigned to linkage groups at a LOD of 4.0, 31 were assigned at a LOD of 3.0, 15 assigned at a LOD of 2.0, and 14 at a LOD of 1.5. Finally, 13 markers were discarded due to conflicting linkage, 3 markers demonstrated linkage to groups at LODs <1.5 but were discarded, and 5 markers were unlinked.

Figure 3.

Figure 3.

Open in a new tab

Linkage map of I. brevicaulis IRRE retrotransposon display markers segregating in IfBC1 hybrids. The first letter of each marker name (a or c) indicates IRRE-A1 and IRRE-C marker loci, respectively. The last four letters in each marker name indicate the EcoRI primer combination used to resolve the locus. Framework marker placements are indicated by a horizontal slash. Placed markers are shown in their most likely interval of location and are indicated by italics. Intermarker recombination distances are shown in Kosambi centimorgans. Markers showing statistically significant transmission ratio distortion are designated by x (α < 0.05) or a solid circle (α < 0.01).

The final I. brevicaulis linkage map consists of 106 framework markers on 22 linkage groups (Figure 3). A total of 180 additional markers were placed on the map but could not be assigned to a single interval at our statistical thresholds (multipoint order support of an LOD at least 2.0).

The number of linkage groups resolved (22) is one greater than the 21 haploid chromosomes reported for I. brevicaulis (Randolph et al. 1961), but is equal to the number of linkage groups resolved in the I. fulva linkage map (discussed above). The number of framework markers per linkage group ranges from 2 to 10, and linkage groups vary in length from 0.0 to 166 cM (Figure 3). The total length of all linkage groups resolved in the I. brevicaulis linkage map is 1108.6 cM. The average marker spacing is 13.0 cM. Estimates of the total map length range from 1708.5 cM, calculated by adding 2s to each of the 22 linkage groups, to 1604.4 cM, calculated using method 4 of Chakravarti et al. (1991). This results in a coverage estimate of 72–74% of the genome within 10 cM of a framework marker. The coefficient of dispersion of 2.80 indicates that markers are clustered.

Little transmission ratio distortion is evident in markers segregating in backcross hybrids between I. fulva and I. brevicaulis:

A minority of If allele markers segregating in IbBC1 hybrids shows deviation from Mendelian expectation. Of the 401 markers in the map, 15.7% show distortion at the α = 0.05 significance level. The majority (71.9%) of these show an overrepresentation of the introgressed If174 allele. From examining the pattern of distorted loci on the linkage map, five map regions (linkage groups 2, 5, 6, 11, and 21) appear to contain putative TRDLs on the basis of our criteria of at least 2 contiguous distorted framework markers (Figure 4A). All but one of these map regions (linkage group 21) are areas of overrepresentation of the introgressed If allele. Only 1 of the 13 markers that were discarded during the mapping process showed statistically significant TRD.

Figure 4.

Figure 4.

Open in a new tab

Observed frequencies of introgressed heterospecific alleles segregating in IbBC1 (A) and IfBC1 (B) hybrids. Each linkage group is represented by a graph and genetic distances are given on the x-axis in Kosambi centimorgans. Only framework marker loci are shown. Frequencies of the introgressed marker alleles are indicated on the y-axis (expected value is 0.50): Frequencies >0.50 indicate an overrepresentation of the introgressed alleles, while frequencies <0.50 indicate an underrepresentation of the introgressed alleles. Marker transmission ratio distortion as evaluated by χ2 tests is indicated by an open circle (nonsignificant), x (α < 0.05), and a solid circle (α < 0.01). For linkage groups encompassing two or more contiguous markers showing significant transmission ratio distortion, the Bayesian posterior probability of a TRDL is given in italics and the posterior distribution of TRDL location is shown as an accompanying histogram (y-axis is the percentage of MCMC runs localizing the TRDL in 5-cM intervals).

A similar minority of the Ib allele markers segregating in the IfBC1 hybrids shows deviation from Mendelian expectation. Of the 286 markers in the map, 15.3% show distortion at the α = 0.05 significance level. As in the I. fulva mapping analysis, the majority (56.8%) of distorted markers incorporated into the I. brevicaulis linkage map show an overrepresentation of the introgressed (Ib72) allele. On the basis of our criteria of at least 2 contiguous distorted framework markers four areas (linkage groups 4, 5, 13, and 17) appear to contain TRDLs (Figure 4B). Three of these (groups 4, 13, and 18) are areas of overrepresentation of the Ib72 allele, while one (group 5) is an area of underrepresentation of the Ib72 allele. Four (19%) of the 21 markers that were not included in the final linkage map showed statistically significant TRD.

The Bayesian MCMC method of Vogl and Xu (2000) was used as an additional method of mapping putative TRDLs. The posterior probability of a TRDL occurring in a given linkage group and the posterior distribution of TRDL positions were estimated from the results of 3000 MCMC iterations. For linkage groups identified as containing putative TRDLs on the basis of χ2 tests (see above), the posterior probabilities for the presence of a TRDL ranged from 0.28 to 0.34 (Figure 4), with an average of 0.31. However, posterior probabilities for the presence of at least one TRDL on linkage groups for which no putative TRDLs were identified were also, on average, 0.31 (data not shown). Examination of the posterior distributions of TRDL locations indicates that the Bayesian MCMC procedure failed to conclusively localize TRDL in correspondence with regions of two or more distorted makers (Figure 4).

DISCUSSION

Genetic mapping of I. fulva and I. brevicaulis using IRRE retrotransposon display markers:

Genetic maps of I. fulva and I. brevicaulis were constructed entirely with markers based on the IRRE LTR retroelement (Kentner et al. 2003). Mapping studies of other species, including barley, peas, and rice, have employed retroelement markers, but these studies incorporated retroelement-based markers into previously developed framework maps (Waugh et al. 1997; Ellis et al. 1998; Manninen et al. 2000; Jiang et al. 2002). This study appears to be the first case of constructing linkage maps de novo using retroelement-based transposon display markers in wild species. Total lengths of the I. fulva and I. brevicaulis maps presented here are estimated at 1966–1980 cM and 1604–1709 cM, respectively, resulting in coverage of 76 and 72% of the I. fulva and I. brevicaulis genomes within 10 cM of a marker. Unfortunately, the homology of linkage groups in the I. fulva and I. brevicaulis maps cannot be established with dominant IRRE retrotransposon markers alone as analyzed here. Both maps had a coefficient of dispersion >1.0, indicating that IRRE transposon display markers are clustered. Clustering and nesting of retrotransposons has been reported in other genetic and physical mapping studies (Waugh et al. 1997; SanMiguel et al. 1998; Kumekawa et al. 1999; Shirasu et al. 2000). However, our results indicate that IRRE transposon display markers provide map coverage adequate for initial genetic and QTL mapping studies. These results demonstrate the utility of transposon display markers for studies of species with few genetic marker resources or with large genomes that can prove intractable for standard marker methods like microsatellites and AFLPs.

The mapping populations were generated from two wild-collected, heterozygous individuals, and this may have had consequences for the construction of the maps. The higher He of 0.59 for Ib72 caused us to have a lower number of Ib72 bands inherited by and segregating from the F1(2) IfBC1 parent, as compared with the number of If174 bands segregating in the IbBC1 hybrids (If174 He = 0.31). This resulted in a smaller data set for construction of the I. brevicaulis linkage map as compared to the I. fulva map (Table 1). The I. brevicaulis map both is shorter and has less coverage than the I. fulva linkage map (1709 cM vs. 1980 cM and 72% vs. 76% coverage), and it includes five linkage groups composed of just two marker loci each. Other genetic mapping studies of wild species used cultivated inbred lines as recurrent parents (Rieseberg et al. 1995; Kim and Rieseberg 1999; Burke et al. 2002) or made inbred lines before initiating the mapping crosses (Fishman et al. 2001). Such approaches are likely to increase the success of mapping projects, but they are not always possible in studies of diverse wild plants such as iris, which takes over a year to begin flowering.

Genomes of both I. fulva and I. brevicaulis appear highly permeable to reciprocal introgression via backcrossing:

Genetic mapping studies provide a means of assessing the number and distribution of genetic factors underlying species boundaries, including genomic regions contributing to hybrid fitness (Rieseberg et al. 1999). In linkage mapping experiments based on interspecific crosses, TRD at marker loci may indicate linkage to alleles conferring reduced or enhanced fertility, viability, or early survival in the hybrid mapping population (Paterson et al. 1991; Bernacchi and Tanksley 1997; Whitkus 1998; Rieseberg et al. 1999; Turelli and Orr 2000; Fishman et al. 2001), although the effects of meiotic drive systems cannot be ruled out. In this study, plants were genotyped after the mapping populations had been cultured in a greenhouse for ∼18 months, meaning that map regions of TRD may reflect genotypic-specific differences in pollen viability or competition, embryo viability, germination rate, or seedling survival and establishment.

A minority of markers in both the I. fulva and the I. brevicaulis map show TRD (∼15% in both maps; Figure 4), implying that little of either species' genome appears resistant to introgression from the other. Each of the maps includes only one map region (linkage group 21 on Figure 4A and linkage group 5 on Figure 4B) in which there appears to be a significantly reduced rate of introgression of heterospecific alleles. The linkage map of I. fulva includes an additional four regions in which the heterospecific alleles are overrepresented, and the linkage map of I. brevicaulis shows three such regions (Figure 4). The proportion of markers showing TRD is lower than is typically seen in interspecific backcross mapping experiments in which, on average, 30% of markers show TRD (Jenczewski et al. 1997), although a considerable range is observed. For example, 30 and 60% of marker loci mapped in interspecific backcrosses between species of Helianthus show TRD (Rieseberg et al. 1995; Kim and Rieseberg 1999). Paterson et al. (1988) reported that 69% of marker loci mapped in an interspecific backcross of Lycopersicon esculentum × (L. esculentum × L. chmielewskii) show TRD, while Bernacchi and Tanksley (1997) reported that 20% of marker loci exhibited TRD in a backcross of L. esculentum × (L. esculentum × L. hirsutum). An interspecific backcross of Oryza sativa × (O. sativa × O. longistamina) reported only 16% distorted marker loci (Causse et al. 1994).

Our χ2 method of identifying map regions of TRD involved multiple tests. However, for both maps, the number of markers showing significant TRD is greater than the number that would be expected by chance alone. Of the If allele markers segregating in IbBC1 hybrids, 30 and 14 framework loci showed TRD at the 0.05 and 0.01 significance levels, respectively, whereas only 7.1 and 1.42 would be expected by chance. Similarly, 13 and 9 of the framework loci in the I. brevicaulis linkage map showed significant TRD at the 0.05 and 0.01 significance levels, respectively, whereas 5.3 and 1.6 would be expected to do so by chance alone.

Results of the Bayesian MCMC procedure of Vogl and Xu (2000) also support the conclusion that few genetic incompatibilities affect introgression between I. fulva and I. brevicaulis and in fact imply fewer species barriers than do the results of the χ2 method. Using a very conservative threshold for the detection of a TRDL (<50% of MCMC iterations resulting in 0 TRDL in a linkage group), only linkage group 18 in the I. fulva genetic map (Figure 4A) was found by the MCMC procedure to contain a TRDL (posterior probability of 0 TRDL = 0.4740). This linkage group did contain two loci that show statistically significant TRD using χ2 tests (Figure 4A), but these markers were not contiguous. However, three of four markers on this linkage group have lower-than-expected frequencies of the introgressed allele, a pattern suggestive of the presence of a TRDL (Figure 4A). Linkage groups that did contain two or more contiguous distorted markers did not have correspondingly higher posterior probabilities for containing TRDLs than linkage groups that did not have contiguous distorted markers (average posterior probabilities for the presence of a TRDL = 0.31 for both). Previous studies employing the Bayesian MCMC procedure of Vogl and Xu (2000) reported generally good congruence between TRDLs mapped using this method and those identified by marker χ2 tests (Fishman et al. 2001; Kuittinen et al. 2004; Myburg et al. 2004). However, these studies examined crosses in which both a greater proportion of markers were distorted and TRD was more severe than reported here in Iris (Fishman et al. 2001; Kuittinen et al. 2004; Myburg et al. 2004). In addition, the MCMC procedure is sensitive to mild deviations in expected allele frequencies, which occurs in many areas of the linkage maps presented here (C. Vogl, personal communication). Determining whether these deviations reflect linkage to genetic factors causing mild TRD would require mapping additional markers to better delimit these areas.

The low occurrence of TRD in the maps presented here is somewhat surprising, given the previous findings of skewed multilocus genotype frequencies indicating high seed abortion rates for certain hybrid classes (Cruzan and Arnold 1994) and the presence of significantly skewed multilocus genotype frequencies in experimental F2 crosses (Burke et al. 1998). The differences in the results of this study and previous work may hinge on the differences in the genotypic classes surveyed and the degree of inbreeding used in the experimental crossing designs. Burke et al. (1998) documented a significant underrepresentation of some multilocus genotypes in outcrossed F2 hybrid seedlings of I. fulva and I. brevicaulis. Genotypes present in F2 hybrids would include those subject to breakdown due to both recessive and dominant-negative epistatic interactions between I. fulva and I. brevicaulis alleles. Theory predicts that F2 hybrids should be subject to more hybrid breakdown than BC1 hybrids (Turelli and Orr 2000). Cruzan and Arnold's (1994) work involved surveying multilocus genotype frequencies at the embryonic stage in a natural hybrid zone population using dominant RAPD markers. It is unknown what the genetic pedigree of Cruzan and Arnold's (1994) plants were: aborted genotypes may have indeed been homozygous for alleles from different species at different loci, leading to hybrid breakdown in viability and the observed skew in multilocus genotype frequencies.

In this study, most of the markers showing TRD do so because of an overrepresentation of introgressed alleles (Figure 4). In contrast to this result, it is generally expected that the introgression of heterospecific alleles should have negative fitness consequences (Rieseberg and Carney 1998), resulting in their occurrence at lower-than-expected frequencies. However, other mapping studies of interspecific crosses have also found areas in which rates of introgression appear to be accelerated by an overrepresentation of heterospecific alleles. In a study of a BC1 between two species of tomato, Bernacchi and Tanksley (1997) reported a total of nine genetic map regions exhibiting TRD, six of which were due to an overrepresentation of the introgressed parent alleles. A study of reciprocal BC1 crosses between two species of Eucalyptus reported that half of the regions of TRD in both maps were due to overrepresentation of introgressed alleles (Myburg et al. 2004), leading these authors to conclude that heterospecific alleles had positive effects on hybrid fitness, which caused rates of introgression to be accelerated in some map regions. Studies of backcross hybrids in Helianthus have also detected genetic map regions in which introgressed alleles are overrepresented, although the majority of TRD is due to underrepresentation of the introgressing alleles (Rieseberg et al. 1995, 1996).

It is important to recognize that the overrepresentation of heterospecific alleles in both hybrid populations presented here could in fact reflect selection against homozygosity in some map regions due to inbreeding depression caused by our experimental design. Our crossing design, in which the same genotypes were used as both grandparents and parents of the BC1 hybrids, was designed to minimize the amount of genetic variation segregating in the mapping populations and thus to maximize the future potential use of these populations for QTL mapping studies. However, this also means that our results might be confounded by inbreeding depression in the BC1 hybrids: map regions in which introgressed parent alleles are overrepresented may be simply due to selection against genotypes that are homozygous for the recurrent parent alleles. In other words, because the fitness effects of introgressed alleles might be modulated by the fitness effects of inbreeding in our study, we may have underestimated the number of genomic regions contributing to species barriers in natural, outcrossing hybrid populations. More crossing experiments would need to be done to evaluate this possibility.

Implications for the evolutionary dynamics of hybridization between I. fulva and I. brevicaulis:

Generalizations based on the results presented here must be made with caution until the completion of additional crossing experiments and map-based investigations of wild hybrid populations (Rieseberg et al. 1999). As in most genetic mapping studies, the results reported here may be specific to the individual genotypes crossed. Further, the fact that IRRE transposon display markers are highly polymorphic in both I. fulva and I. brevicaulis populations (reported in Kentner et al. 2003 at 45 and 63% for IRRE-A1 and IRRE-C in I. fulva and at 84 and 82% for IRRE1-A1 and IRRE1-C in I. brevicaulis) will present a challenge for the extension of these results to studies of wild hybrid populations until the conversion of mapped loci to species-specific codominant markers can be completed. However, given the potential for natural hybridization and introgression to contribute to adaptation and speciation, determining which portions of hybridizing plant genomes are permeable to introgression and which are resistant is important for understanding the possible outcomes of hybridization (Rieseberg et al. 1995, 2000; Arnold 2004a,b).

Overall, the results of both methods employed to analyze TRD suggest that little of either species' genome should show nonneutral introgression in natural hybrid zones due to intrinsic genetic incompatibilities alone. Results of the χ2 method were perhaps somewhat more conclusive in that this analysis identified a small number of genomic regions of TRD, providing a baseline for future investigations of introgression in natural populations. Actual patterns of introgression in hybrid zone populations of Louisiana Iris species are affected by a complex cascade of factors. These include ecological setting, timing of flowering, floral traits, pollinator behavior, gamete competition, and hybrid viability and fertility (Arnold et al. 1993; Carney et al. 1994; Emms et al. 1996; Burke et al. 1998; Emms and Arnold 2000; Wesselingh and Arnold 2000; Johnston et al. 2001a, 2003). Since all these factors interact, genetic covariance between numerous traits might be important in preventing or facilitating gene flow between I. fulva and I. brevicaulis in natural settings. Future investigations will examine the presence of genetic covariance among hybrid fitness, ecological traits, and floral morphology, as well as their effects on fitness in experimental hybrid populations.

Acknowledgments

We thank Rodney Mauricio for providing invaluable advice throughout the course of this project. Michael Boyd and Andrew Tull provided expert plant care with the assistance of Rebecca Koopman, Erinn Morgan, and Nicole Schell. Eric Anderson assisted in implementation of the Bayesian MCMC analysis. This manuscript benefited from critical review by Noland Martin, James L. Hamrick, Gina Baucom, Rodney Mauricio, Matt Campbell, Scott Cornman, and two anonymous reviewers. M.L.A. was supported by National Science Foundation (NSF) grants DEB-0074159 and DEB-0345123. A.B. was supported by an NSF Predoctoral Fellowship, the National Institutes of Health (NIH), and a University of Georgia Dissertation Completion Award. S.R.W. was supported by the NSF and the NIH.

References

  1. Anderson, E., 1948. Hybridization of the habitat. Evolution 2: 1–9. [Google Scholar]
  2. Anderson, E., 1949. Introgressive Hybridization. John Wiley & Sons, New York.
  3. Anttila, C. K., R. A. King, C. Ferris, D. R. Ayres and D. R. Strong, 2000. Reciprocal hybrid formation of Spartina in San Francisco Bay. Mol. Ecol. 9: 765–770. [DOI] [PubMed] [Google Scholar]
  4. Arnold, M. L., 1997. Natural Hybridization and Evolution. Oxford University Press, New York.
  5. Arnold, M. L., 2004. a Natural hybridization and the evolution of domesticated, pest and disease organisms. Mol. Ecol. 13: 997–1007. [DOI] [PubMed] [Google Scholar]
  6. Arnold, M. L., 2004. b Transfer and origin of adaptations through natural hybridization: Were Anderson and Stebbins right? Plant Cell 16: 562–570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Arnold, M. L., J. J. Robinson, C. M. Buckner and B. D. Bennet, 1992. Pollen dispersal and interspecific gene flow in Louisiana irises. Heredity 68: 399–404. [Google Scholar]
  8. Arnold, M. L., J. L. Hamrick and B. D. Bennett, 1993. Interspecific pollen competition and reproductive isolation in Iris. J. Hered. 84: 13–16. [Google Scholar]
  9. Arnold, M. L., E. K. Kentner, J. A. Johnston, S. Cornman and A. C. Bouck, 2001. Natural hybridisation and fitness. Taxon 50: 93–104. [Google Scholar]
  10. Barbash, D. A., P. Awadalla and A. M. Tarone, 2004. Functional divergence caused by ancient positive selection of a Drosophila hybrid incompatibility locus. PloS Biol. 2: 839–848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Barton, N. H., and G. M. Hewitt, 1985. Analysis of hybrid zones. Annu. Rev. Ecol. Syst. 16: 113–148. [Google Scholar]
  12. Bateson, W., 1909. Heredity and variation in modern lights, pp. 85–101 in Darwin and Modern Science, edited by A. C. Steward. Cambridge University Press, Cambridge, UK.
  13. Bernacchi, D., and S. D. Tanksley, 1997. An interspecific backcross of Lycopersicon esculentum × L. hirsutum: linkage analysis and a QTL study of sexual compatibility factors and floral traits. Genetics 147: 861–877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Blair, W. F., 1955. Mating call and stage of speciation in the Microhyla olivacea-M. carolinensis complex. Evolution 9: 469–480. [Google Scholar]
  15. Bradshaw, H. D., S. M. Wilbert, K. G. Otto and D. W. Schemske, 1995. Genetic-mapping of floral traits associated with reproductive isolation in monkeyflowers (Mimulus). Nature 376: 762–765. [Google Scholar]
  16. Bradshaw, H. D., K. G. Otto, B. E. Frewen, J. K. McKay and D. W. Schemske, 1998. Quantitative trait loci affecting differences in floral morphology between two species of monkeyflower (Mimulus). Genetics 149: 367–382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Buerkle, C. A., and L. H. Rieseberg, 2001. Low intraspecific variation for genomic isolation between hybridizing sunflower species. Evolution 55: 684–691. [DOI] [PubMed] [Google Scholar]
  18. Burke, J. M., T. J. Voss and M. L. Arnold, 1998. Genetic interactions and natural selection in Louisiana Iris hybrids. Evolution 52: 1304–1310. [DOI] [PubMed] [Google Scholar]
  19. Burke, J. M., S. Tang, S. J. Knapp and L. H. Rieseberg, 2002. Genetic analysis of sunflower domestication. Genetics 161: 1257–1267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Carney, S. E., M. B. Cruzan and M. L. Arnold, 1994. Reproductive interactions between hybridizing irises—analyses of pollen-tube growth and fertilization success. Am. J. Bot. 81: 1169–1175. [Google Scholar]
  21. Casa, A. M., C. Brouwer, A. Nagel, L. J. Wang, Q. Zhang et al., 2000. The MITE family Heartbreaker (Hbr): molecular markers in maize. Proc. Natl. Acad. Sci. USA 97: 10083–10089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Causse, M. A., T. M. Fulton, Y. G. Cho, S. N. Ahn, J. Chunwongse et al., 1994. Saturated molecular map of the rice genome based on an interspecific backcross population. Genetics 138: 1251–1274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Cervera, M. T., V. Storme, B. Ivens, J. Gusmao, B. H. Liu et al., 2001. Dense genetic linkage maps of three Populus species (Populus deltoides, P. nigra and P. trichocarpa) based on AFLP and microsatellite markers. Genetics 158: 787–809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Chakravarti, A., L. K. Lasher and J. E. Reefer, 1991. A maximum-likelihood method for estimating genome length using genetic-linkage data. Genetics 128: 175–182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Cruzan, M. B., and M. L. Arnold, 1993. Ecological and genetic associations in an iris hybrid zone. Evolution 47: 1432–1445. [DOI] [PubMed] [Google Scholar]
  26. Cruzan, M. B., and M. L. Arnold, 1994. Assortative mating and natural-selection in an iris hybrid zone. Evolution 48: 1946–1958. [DOI] [PubMed] [Google Scholar]
  27. Dobzhansky, T. H., 1936. Studies in hybrid sterility. II. Localization of sterility factors in Drosophila pseudoobscura hybrids. Genetics 21: 113–135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Dobzhansky, T. H., 1937. Genetics and the Origin of Species. Columbia University Press, New York.
  29. Dobzhansky, T., 1940. Speciation as a stage in evolutionary divergence. Am. Nat. 74: 312–321. [Google Scholar]
  30. Doebley, J., A. Stec and C. Gustus, 1995. Teosinte branched1 and the origin of maize: evidence for epistasis and the evolution of dominance. Genetics 141: 333–346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Doyle, J. J., and J. L. Doyle, 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19: 11–15. [Google Scholar]
  32. Ellis, T. H. N., S. J. Poyser, M. R. Knox, A. V. Vershinin and M. J. Ambrose, 1998. Polymorphism of insertion sites of Ty1-copia class retrotransposons and its use for linkage and diversity analysis in pea. Mol. Gen. Genet. 260: 9–19. [DOI] [PubMed] [Google Scholar]
  33. Emms, S. K., and M. L. Arnold, 2000. Site-to-site differences in pollinator visitation patterns in a Louisiana iris hybrid zone. Oikos 91: 568–578. [Google Scholar]
  34. Emms, S. K., S. A. Hodges and M. L. Arnold, 1996. Pollen-tube competition, siring success, and consistent asymmetric hybridization in Louisiana irises. Evolution 50: 2201–2206. [DOI] [PubMed] [Google Scholar]
  35. Fishman, L., and J. H. Willis, 2001. Evidence for Dobzhansky-Muller incompatibilities contributing to the sterility of hybrids between Mimulus guttatus and M. nasutus. Evolution 55: 1932–1942. [DOI] [PubMed] [Google Scholar]
  36. Fishman, L., A. J. Kelly, E. Morgan and J. H. Willis, 2001. A genetic map in the Mimulus guttatus species complex reveals transmission ratio distortion due to heterospecific interactions. Genetics 159: 1701–1716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Fishman, L., A. J. Kelly and J. H. Willis, 2002. Minor quantitative trait loci underlie floral traits associated with mating system divergence in Mimulus. Evolution 56: 2138–2155. [DOI] [PubMed] [Google Scholar]
  38. Flavell, A. J., M. R. Knox, S. R. Pearce and T. H. N. Ellis, 1998. Retrotransposon-based insertion polymorphisms (RBIP) for high throughput marker analysis. Plant J. 16: 643–650. [DOI] [PubMed] [Google Scholar]
  39. Grant, P. R., and B. R. Grant, 2002. Unpredictable evolution in a 30-year study of Darwin's finches. Science 296: 707–711. [DOI] [PubMed] [Google Scholar]
  40. Harrison, R. G., 1990. Hybrid zones: windows on evolutionary process, pp. 69–128 in Oxford Surveys in Evolutionary Biology, edited by D. J. Futuyma and J. Antonovics. Oxford University Press, New York.
  41. Harushima, Y., M. Nakagahra, M. Yano, T. Sasaki and N. Kurata, 2001. A genome-wide survey of reproductive barriers in an intraspecific hybrid. Genetics 159: 883–892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Harushima, Y., M. Nakagahra, M. Yano, T. Sasaki and N. Kurata, 2002. Diverse variation of reproductive barriers in three intraspecific rice crosses. Genetics 160: 313–322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Howard, D. J., 1993. Reinforcement: origins, dynamics, and the fate of an evolutionary hypothesis, pp. 46–69 in Hybrid Zones and Evolutionary Process, edited by R. G. Harrison. Oxford University Press, New York.
  44. Huxel, G. R., 1999. Rapid displacement of native species by invasive species: effects of hybridization. Biol. Conserv. 89: 143–152. [Google Scholar]
  45. Jenczewski, E., M. Gherardi, I. Bonnin, J. M. Prosperi, I. Olivieri et al., 1997. Insight on segregation distortions in two intraspecific crosses between annual species of Medicago (Leguminosae). Theor. Appl. Genet. 94: 682–691. [Google Scholar]
  46. Jiang, C. X., P. W. Chee, X. Draye, P. L. Morrell, C. W. Smith et al., 2000. Multilocus interactions restrict gene introgression in interspecific populations of polyploid Gossypium (cotton). Evolution 54: 798–814. [DOI] [PubMed] [Google Scholar]
  47. Jiang, N., Z. Bao, S. Temnykh, Z. Cheng, J. Jiang et al., 2002. Dasheng: a recently amplified nonautonomous long terminal repeat element that is a major component of pericentromeric regions in rice. Genetics 161: 1293–1305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Johnston, J. A., D. J. Grise, L. A. Donovan and M. L. Arnold, 2001. a Environment-dependent performance and fitness of Iris brevicaulis, I. fulva (Iridaceae), and hybrids. Am. J. Bot. 88: 933–938. [PubMed] [Google Scholar]
  49. Johnston, J. A., R. A. Wesselingh, A. C. Bouck, L. A. Donovan and M. L. Arnold, 2001. b Intimately linked or hardly speaking? The relationship between genotype and environmental gradients in a Louisiana Iris hybrid population. Mol. Ecol. 10: 673–681. [DOI] [PubMed] [Google Scholar]
  50. Johnston, J. A., M. L. Arnold and L. A. Donovan, 2003. High hybrid fitness at seed and seedling life history stages in Louisiana irises. J. Ecol. 91: 438–446. [Google Scholar]
  51. Kalendar, R., T. Grob, M. Regina, A. Suoniemi and A. Schulman, 1999. IRAP and REMAP: two new retrotransposon-based DNA fingerprinting techniques. Theor. Appl. Genet. 98: 704–711. [Google Scholar]
  52. Kentner, E. K., M. L. Arnold and S. R. Wessler, 2003. Characterization of high-copy-number retrotransposons from the large genomes of the Louisiana Iris species and their use as molecular markers. Genetics 164: 685–697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Key, K. H. L., 1968. Concept of stasipatric speciation. Syst. Zool. 17: 14. [Google Scholar]
  54. Kim, S. C., and L. H. Rieseberg, 1999. Genetic architecture of species differences in annual sunflowers: implications for adaptive trait introgression. Genetics 153: 965–977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Kinlaw, C. S., and D. B. Neale, 1997. Complex gene families in pine genomes. Trends Plant Sci. 2: 356–359. [Google Scholar]
  56. Kuittinen, H., A. A. de Haan, C. Vogl, S. Oikarinen, J. Leppala et al., 2004. Comparing the linkage maps of the close relatives Arabidopsis lyrata and A. thaliana. Genetics 168: 1575–1584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Kumekawa, N., H. Ohtsubo, T. Horiuchi and E. Ohtsubo, 1999. Identification and characterization of novel retrotransposons of the gypsy type in rice. Mol. Gen. Genet. 260: 593–602. [DOI] [PubMed] [Google Scholar]
  58. Lander, E., J. Abrahamson, A. Barlow, M. Daly, S. Lincoln et al., 1987. Mapmaker: a computer package for constructing genetic-linkage maps. Cytogenet. Cell Genet. 46: 642. [DOI] [PubMed] [Google Scholar]
  59. Lin, J. Z., and K. Ritland, 1997. Quantitative trait loci differentiating the outbreeding Mimulus guttatus from the inbreeding M. platycalyx. Genetics 146: 1115–1121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Lincoln, S., M. Daly and E. Lander, 1992. Constructing genetic maps with MAPMAKER/EXP 3.0. Whitehead Institute Technical Report. Whitehead Institute, Cambridge, MA.
  61. Manninen, O., R. Kalendar, J. Robinson and A. H. Schulman, 2000. Application of BARE-1 retrotransposon markers to the mapping of a major resistance gene for net blotch in barley. Mol. Gen. Genet. 264: 325–334. [DOI] [PubMed] [Google Scholar]
  62. Marshall, J. L., M. L. Arnold and D. J. Howard, 2002. Reinforcement: the road not taken. Trends Ecol. Evol. 17: 558–563. [Google Scholar]
  63. Moyle, L. C., and E. B. Graham, 2005. Genetics of hybrid incompatibility between Lycopersicon esculentum and L. hirsutum. Genetics 169: 355–373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Muller, H. J., 1940. Bearings of the Drosophila work on systematics, pp. 185–268 in The New Systematics, edited by J. Huxley. Clarendon Press, Oxford, UK.
  65. Myburg, A. A., C. Vogl, A. R. Griffin, R. R. Sederoff and R. W. Whetten, 2004. Genetics of postzygotic isolation in eucalyptus: whole-genome analysis of barriers to introgression in a wide interspecific cross of Eucalyptus grandis and E. globulus. Genetics 166: 1405–1418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Navarro, A., and N. H. Barton, 2003. Chromosomal speciation and molecular divergence: accelerated evolution in rearranged chromosomes. Science 300: 321–324. [DOI] [PubMed] [Google Scholar]
  67. Orr, H. A., 1995. The population genetics of speciation: the evolution of hybrid incompatibilities. Genetics 139: 1805–1813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Orr, H. A., and M. Turelli, 2001. The evolution of postzygotic isolation: accumulating Dobzhansky-Muller incompatibilities. Evolution 55: 1085–1094. [DOI] [PubMed] [Google Scholar]
  69. Paterson, A. H., E. S. Lander, J. D. Hewitt, S. Peterson, S. E. Lincoln et al., 1988. Resolution of a quantitative trait into Mendelian factors by using a complete linkage map of restriction fragment length polymorphisms. Nature 335: 721–726. [DOI] [PubMed] [Google Scholar]
  70. Paterson, A. H., S. Damon, J. D. Hewitt, D. Zamir, H. D. Rabinowitch et al., 1991. Mendelian factors underlying quantitative traits in tomato: comparison across species, generations and environments. Genetics 127: 181–197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Pfeiffer, A., A. M. Olivieri and M. Morgante, 1997. Identification and characterization of microsatellites in Norway spruce (Picea abies K). Genome 40: 411–419. [DOI] [PubMed] [Google Scholar]
  72. Presgraves, D. C., 2003. A fine-scale genetic analysis of hybrid incompatibilities in Drosophila. Genetics 163: 955–972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Presgraves, D. C., L. Balagopalan, S. M. Abmayr and H. A. Orr, 2003. Adaptive evolution drives divergence of a hybrid inviability gene between two species of Drosophila. Nature 423: 715–719. [DOI] [PubMed] [Google Scholar]
  74. Randolph, L. F., J. Mitra and I. S. Nelson, 1961. Cytotaxonomic studies of Louisiana irises. Bot. Gaz. 123: 126–131. [Google Scholar]
  75. Randolph, L. F., J. Mitra and I. S. Nelson, 1967. Negative evidence of introgression affecting the stability of Louisiana iris species. Cornell University Agricultural Experimental Station Memoir 398: 1–56. [Google Scholar]
  76. Remington, D. L., R. W. Whetten, B. H. Liu and D. M. O'Malley, 1999. Construction of an AFLP genetic map with nearly complete genome coverage in Pinus taeda. Theor. Appl. Genet. 98: 1279–1292. [DOI] [PubMed] [Google Scholar]
  77. Rhymer, J. M., and D. Simberloff, 1996. Extinction by hybridization and introgression. Annu. Rev. Ecol. Syst. 27: 83–109. [Google Scholar]
  78. Rieseberg, L. H., and S. E. Carney, 1998. Plant hybridization. New Phytol. 140: 599–624. [DOI] [PubMed] [Google Scholar]
  79. Rieseberg, L. H., C. R. Linder and G. J. Seiler, 1995. Chromosomal and genic barriers to introgression in Helianthus. Genetics 141: 1163–1171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  80. Rieseberg, L. H., B. Sinervo, C. R. Linder, M. C. Ungerer and D. M. Arias, 1996. Role of gene interactions in hybrid speciation: evidence from ancient and experimental hybrids. Science 272: 741–745. [DOI] [PubMed] [Google Scholar]
  81. Rieseberg, L. H., J. Whitton and K. Gardner, 1999. Hybrid zones and the genetic architecture of a barrier to gene flow between two sunflower species. Genetics 152: 713–727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. Rieseberg, L. H., S. J. E. Baird and K. A. Gardner, 2000. Hybridization, introgression, and linkage evolution. Plant Mol. Biol. 2: 205–224. [PubMed] [Google Scholar]
  83. Rieseberg, L. H., O. Raymond, D. M. Rosenthal, Z. Lai, K. Livingstone et al., 2003. Major ecological transitions in wild sunflowers facilitated by hybridization. Science 301: 1211–1216. [DOI] [PubMed] [Google Scholar]
  84. SanMiguel, P., B. S. Gaut, A. Tikhonov, Y. Nakajima and J. L. Bennetzen, 1998. The paleontology of intergene retrotransposons of maize. Nat. Genet. 20: 43–45. [DOI] [PubMed] [Google Scholar]
  85. Seehausen, O., 2004. Hybridization and adaptive radiation. Trends Ecol. Evol. 19: 198–207. [DOI] [PubMed] [Google Scholar]
  86. Shirasu, K., A. H. Schulman, T. Lahaye and P. Schulze-Lefert, 2000. A contiguous 66-kb barley DNA sequence provides evidence for reversible genome expansion. Genome Res. 10: 908–915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  87. Small, J. K., and E. J. Alexander, 1931. Botanical interpretations of the iridaceous plants of the Gulf States. Contrib. NY Bot. Gard. 327: 325–357. [Google Scholar]
  88. Sokal, R. R., and F. J. Rohlf, 1995. Biometry. W. H. Freeman, New York.
  89. Soltis, D. E., P. S. Soltis, T. G. Collier and M. L. Edgerton, 1991. Chloroplast DNA variation within and among genera of the Heuchera group (Saxifragaceae): evidence for chloroplast transfer and paraphyly. Am. J. Bot. 78: 1091–1112. [Google Scholar]
  90. Turelli, M., and H. A. Orr, 2000. Dominance, epistasis and the genetics of postzygotic isolation. Genetics 154: 1663–1679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  91. Van den Broeck, D., T. Maes, M. Sauer, J. Zethof, P. De Keukeleire et al., 1998. Transposon display identifies individual transposable elements in high copy number lines. Plant J. 13: 121–129. [DOI] [PubMed] [Google Scholar]
  92. Viosca, Jr., P., 1935. The irises of southeastern Louisiana, a taxonomic and ecological interpretation. Bull. Am. Iris Soc. 57: 3–56. [Google Scholar]
  93. Vogl, C., and S. Z. Xu, 2000. Multipoint mapping of viability and segregation distorting loci using molecular markers. Genetics 155: 1439–1447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  94. Vos, P., R. Hodgers, M. Bleeker, M. Reijans, T. Vandelee et al., 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23: 4407–4414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  95. Waugh, R., K. McLean, A. J. Flavell, S. R. Pearce, A. Kumar et al., 1997. Genetic distribution of Bare-1-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP). Mol. Gen. Genet. 253: 687–694. [DOI] [PubMed] [Google Scholar]
  96. Wesselingh, R. A., and M. L. Arnold, 2000. Pollinator behaviour and the evolution of Louisiana Iris hybrid zones. J. Evol. Biol. 13: 171–180. [Google Scholar]
  97. Whitkus, R., 1998. Genetics of adaptive radiation in Hawaiian and Cook Islands species of Tetramolopium (Asteraceae). II. Genetic linkage map and its implications for interspecific breeding barriers. Genetics 150: 1209–1216. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES