Cosmid DNA:

A set of 20 cosmids provided by C. Fauron (Fauron and Havlik 1988) that sequentially cover the entire NB mitochondrial genome (Clifton et al. 2004) was used as mtDNA probes, either individually or in a 19-cosmid mix. Chloramphenicol was added to bacterial cultures that had been grown overnight, and the cosmids were allowed to amplify for an additional 6 hr. Cosmid DNA was isolated from each culture using the QIAGEN (Valencia, CA) Plasmid Maxi-prep protocol.

Karyotyping probes:

A standardized “cocktail” of eight different repeated DNA sequences (Table 1) was used for karyotyping (Kato et al. 2004). The probes used were labeled by nick translation with coumarin-5-dUTP or Cascade Blue-7-dUTP, fluorescein isothiocyanate-12-dUTP (FITC) or Alexa Fluor 488-5-dUTP and Cyanine 5-dUTP (Cy5).

Labeling of cosmid and karyotyping probes:

The mtDNA-containing cosmids were labeled with Texas red-5-dCTP. The optimized nick translation protocol of Kato et al. (2006) was used with the following specifications. For 5 μg DNA (25 μl, 200 ng/μl), the nick translation mix included 1 μl 100 milliunits/μl DNase (2 μl for 19-cosmid mix), 20 μl 10 units/μl DNA polymerase I (Texas red, Cy5), 5 μl 10× nick translation buffer, 5 μl nonlabeled dAGC mix (2 mm, karyotyping probes) or dATG mix (2 mm, mtDNA probes), and 1.25 μl labeled 1 mm dUTP (karyotyping probes) or dCTP (mtDNA probes). After labeling, each of the Texas red- and Cy5-labeled probes were purified on Bio-gel P-60 columns (Kato et al. 2006). After discarding two washes (50 and 350 μl), eluates from 2–3 additional 350 μl 1× TE washes (yielding 2–5 μg DNA) were collected. The labeled DNA was ethanol precipitated, resuspended in 2× SSC/1× TE (TE, pH 8.0), and stored at −20°. Individual mtDNA-containing cosmid probes were resuspended at a final concentration of 100 ng/μl in 2× SSC/1× TE. The 19-cosmid mix probe was at a final concentration of 400 ng/μl. For the nick translation reaction with other fluorochromes (coumarin, Cascade Blue, FITC, and Alexa Fluor 488) 6.25 μl DNA polymerase I (10 units/μl) was used in each reaction. These probes were not column purified but were ethanol precipitated immediately, dissolved in the 2× SSC/1× TE buffer, and stored at −20°.

Preparation of root tip chromosome spreads:

The protocol for the preparation of root tip mitotic metaphase chromosome spreads was performed as described (Kato et al. 2006) with the following modifications. Root tips were not incubated on ice during enzymatic digestion. After dispersing the cells in 70% ethanol, the cells were pelleted by centrifuging for 10–20 sec instead of 2 min. After dropping the cell suspension on slides, the slides were UV crosslinked (120-5 mJ/cm²) and fixed with 10% formaldehyde for 10 min, washed with 100% ethanol, and allowed to dry. Additional modifications to the Kato et al. (2006) protocol were used for the comparisons among the B73 (B), B73 (C), W23-B, and W23 A × B hybrid lines. The root tips were washed in 100% ethanol three times (without a 70% ethanol treatment) and then cells were dispersed by agitation in acetic acid. These samples were not subjected to formaldehyde fixation.

Denaturation protocol:

The procedure for the denaturation of probe and chromosomal DNA separately was the same as described in Kato et al. (2004) with minor modifications. Briefly, after formaldehyde treatment of the slides, 5 μl of 2× SSC/1× TE/salmon sperm DNA (1 μg/μl) was dropped onto the center of the cells and a plastic coverslip applied. The probe was prepared by mixing the various components in a microcentrifuge tube to a final volume of 5 μl. When the 19-cosmid mix was used, the probe for each slide consisted of 2.5 μl 19-cosmid mix, 1.7 μl karyotyping mix listed in Table 1, and 0.8 μl 2× SSC/1× TE. For individual cosmids, the probe for each slide consisted of 1 μl of the individually labeled cosmid and 4 μl of karyotyping mix (with 2× SSC/1× TE). Each slide was incubated at 55° for 3–16 hr in a humid chamber. After hybridization, the slide was washed with 2× SSC at room temperature for 5 min, followed by a wash with 2× SSC at 55° for 20 min in Coplin jars. The slide was mounted with Vectashield (Vector Laboratories, Burlingame, CA) containing 4′,6-diamidino-2-phenylindole (DAPI). The DAPI was diluted to 1/20 strength using Vectashield. Vectashield with no DAPI was used with Cascade Blue probes because DAPI interferes with the probe signal. Slides were stored at −20°.

Image capture and data processing of FISH images:

The protocol for capturing and processing FISH images was as previously described (Kato et al. 2004, 2006) with modifications. Color assignments were as follows: blue (coumarin or Cascade Blue), green (FITC or Alexa Fluor 488), red (Cy5), and white (Texas red). Using Adobe Photoshop, background was reduced by subtracting an image from an empty area of the slide taken at approximately the same exposures for each channel.

Analysis of NUMTs using individual mtDNA-containing cosmids:

The maize NB mitochondrial genome has been sequenced from a normal male-fertile B37 line and consists of 569,630 bp (Clifton et al. 2004). A set of numbered cosmid clones (Fauron and Havlik 1988) covering the whole NB genome on 20 overlapping mtDNA fragments (averaging ∼37 kb each, Figure 1a) was used to make FISH probes. It should be noted that there are two large insertions of cpDNA in the NB mitochondrial genome, as well as multiple, smaller insertions (Stern and Lonsdale 1982; Lonsdale et al. 1983; Clifton et al. 2004). Cosmid 13 (∼39.5 kb) contains the two largest chloroplast DNA insertions (Figure 1a) of 12.6 kb and 4.1 kb (comprising ∼34% of the cosmid). Thus, we expected the mtDNA from cosmid 13 to identify some of the cpDNA as well as mtDNA insertions in nuclear chromosomes.

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Figure 1.—

MtDNA segments hybridized to B73 chromosomes. (a) Linear map of the NB maize mitochondrial genome with cosmid locations. A linearized version of the 569,630 bp NB maize mitochondrial genome is shown (Clifton et al. 2004; condensed from Allen et al. 2007) with the cosmid positions outlined. Mitochondrial genes, rRNAs, and tRNAs (half-length lines) are represented by vertical lines. Repeats are indicated by colored arrows. The cpDNA insertions are indicated by green rectangles. A scale in kilobases is shown along the bottom. (b) Individual mtDNA-containing cosmids hybridized to B73 chromosomes. Individual cosmid probes containing mtDNA were labeled with Texas red and hybridized to B73 chromosomes. The white arrowheads mark mtDNA insertion sites and the green arrowheads mark potential cpDNA insertion sites. The eight mix of karyotyping probes (Kato et al. 2004) was used to identify each chromosome. Only the layer with the Texas red-labeled mitochondrial probes is shown. The marked mtDNA insertion sites were identified on >88% of individual chromosomes examined.

The DNA from each of the 20 cosmids was individually labeled with Texas red and hybridized to B73 mitotic metaphase chromosomes to locate NUMTs (Figure 1b). The B73 inbred line was chosen for this analysis because its nuclear genome is currently being sequenced (Bennetzen et al. 2001) and it has the NB mitochondrial genotype.

Individual mtDNA insertion sites were determined by examining multiple chromosome preparations (at least 11; see supplemental Table 1 at http://www.genetics.org/supplemental/). Due to hybridization of the mtDNA-containing cosmids with organelles present on the chromosome spreads and, less significantly, to chromosomal twisting, it is sometimes difficult to identify a specific NUMT in every case. The mtDNA insertions we have indicated in Figure 1b were consistently present. The minimum target size documented using FISH on maize chromosomes is ∼3 kb (Yu et al. 2007). Thus, the insertions detected in this study are larger than this lower limit.

The mtDNA contained in the individual cosmids identified a total of 58 insertion sites within the B73 chromosomes (Figure 1b). When all of the NUMTs were compared, it was determined that there was a minimum of 21 unique mtDNA insertion sites. The NUMTs were arbitrarily classified by their position on the chromosome: proximal (near centromeres), distal (near telomeres), and interstitial (between the centromere and a telomere). Of the 58 total sites, 32 (55.2%) were proximal and 10 (17.2%) were distal. Most of the cosmids (14 of the 20) identified a specific site located proximally on the long arm of chromosome 9. This insertion apparently includes a large region of mtDNA that is colinear in the mitochondrial genome (contained in cosmids 20, and 1–10) and which could be present on a single mitochondrial subgenome. However, our experiments cannot distinguish whether the mtDNA organization is preserved in the NUMT.

After comparing the B73 NUMTs to the mitochondrial genome map (Figure 1a) (Clifton et al. 2004), no preference was seen for insertions of gene-rich vs. gene-poor regions of mtDNA. No visible NUMTs were identified using cosmid 11, which contains both gene-rich and gene-poor regions (Figure 1b). The mtDNA contained in cosmid 11 is ∼40 kb, of which ∼9.7 kb does not overlap cosmids 10 and 12 (Figure 1a). The region of overlap with cosmid 10 is relatively gene rich, containing nad5 exons 3–5, rps12, nad3, nad1 exon 5, mat-r, and 283 bp of rps1. Detectable NUMTs did include regions of gene-poor mtDNA. For example, cosmid 8 includes ∼38 kb of mtDNA, with ∼10.7 kb not overlapping cosmids 7 and 9 (Figure 1a). It contains only two known genes: ccmC (723 bp) and nad5 exons 1–2 (2316 bp) (Clifton et al. 2004). This cosmid detects two mtDNA insertions, located proximally on 2L and 9L (Figure 1b). Cosmid 15 contains ∼38 kb mtDNA, with ∼32.3 kb not overlapping cosmids 14 and 16. It includes only one gene: rrn26 (3552 bp) (Clifton et al. 2004). The mtDNA within cosmid 15 detects three NUMTs—in 4L proximal, 5L interstitial, and 9S distal. Collectively, these data suggest that gene content does not affect which regions of mtDNA are inserted into the nuclear genome.

Cosmid 13 identified more apparent NUMTs in the B73 nuclear genome than any other cosmid (Figure 1b). However, because the mtDNA contained within this cosmid includes large segments of integrated cpDNA, we attempted to infer which insertion sites could be due to cpDNA. We compared the sites identified with cosmid 13 to those detected using the other cosmids. The two NUMTs on chromosome 4 and one on chromosome 3 were identified by other cosmids, indicating that they were likely mtDNA insertion sites. The insertion on chromosome 2L matches one identified only by the overlapping cosmid 14, which contains 4.1 kb of cpDNA, indicating that this site is potentially a NUPT. A second site identified by cosmid 14 on 4L does appear to be a NUMT because the same site was detected by other cosmids. The remaining seven sites identified by cosmid 13 are potentially cpDNA insertions because no other mtDNA cosmids hybridize to those locations. However, our data set does not allow us to determine conclusively whether the insertions originated from mitochondria or plastid DNA.

If we exclude the sites identified by hybridization with cosmid 13 that potentially represent cpDNA insertions, a total of 51 different mtDNA insertion sites were detected in our FISH analyses. Of the 51 mtDNA insertion sites, 29 (60.4%) were classified as proximal and 9 (18.8%) as distal. When the relative positions of all sites identified by all of the cosmids were compared, a minimum of 15 unique NUMT locations were found to exist in B73. Excluding cosmid 13 from the analyses, NUMTs were not detected on chromosomes 1, 8, or 10 in B73.

Analysis of NUMTs in 10 maize inbred lines:

To assess variability for NUMTs in maize, mtDNA insertions were identified in a set of 10 maize inbred lines using the 19-cosmid mix probe labeled with the most sensitive fluorochrome in our system, Texas red (Figure 2). Cosmid 13 was excluded from the cosmid mix because, as noted above, it carries a region of mtDNA that includes large insertions of cpDNA. The karyotypes of the 10 inbred lines had been previously characterized using FISH with a cocktail of repetitive DNA probes (Kato et al. 2004). A karyotyping cocktail (see materials and methods) was included in the hybridization with the 19-cosmid mix in order to identify the chromosomes of the different inbred lines.

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Figure 2.—

MtDNA insertion sites in the chromosomes of 10 maize inbred lines. The karyotypes of 10 different maize inbred lines are shown. Sites of mtDNA hybridization on the chromosomes, probed with the 19-cosmid mix of mtDNA, are indicated by arrowheads. The eight mix of karyotyping probes was used to identify the chromosomes. The marked mtDNA insertion sites were identified on >88% of the chromosomes examined.

The numbers and locations of the mtDNA hybridization sites on chromosomes varied greatly among the maize inbred lines (Figure 2; see supplemental Table 2 at http://www.genetics.org/supplemental/ for the numbers of chromosomes examined). The number of detected NUMTs ranged from 9 in A188 and A632 to 19 in Oh43. Several of these NUMTs were observed at proximal and distal locations on chromosomes (Table 2). When all the insertions are compared (Figure 2), only one on 2S proximal appeared to be shared among all 10 lines examined. However, due to the limitations inherent in analyzing highly condensed metaphase chromosomes, it is possible that the 2S proximal signals in different lines are at different positions.

The B73 mtDNA insertion sites identified using the 19-cosmid mix (Figure 2) were compared to those detected by the individual mtDNA-containing cosmid probes (Figure 1b). Using the 19-cosmid mix, 13 distinct mtDNA insertion sites were detected in B73. There were two fewer NUMTs identified using the 19-cosmid mix than with the individual mtDNA-containing cosmid probes; they were located on 7S (using cosmid 1) and on 5L (using cosmid 6). This difference in detection ability might be due to the fact that the individual cosmid mtDNA probes contained a larger amount of mtDNA per cosmid than the 19-cosmid mix. The same concentration of mtDNA per cosmid could not be used in the 19-cosmid mix because at high concentrations (>400 ng/μl), the labeled probe DNA becomes very difficult to dissolve. The final concentration of each individual cosmid in the 19-cosmid mix probe cocktail was approximately half the concentration used when individual cosmids were applied.

One additional B73 mtDNA insertion site was identified on 1S using the 19-cosmid mix. It was not observed using individual mtDNA-containing cosmid probes. We hypothesize that this location contains portions of mtDNA from across the mitochondrial genome (contained in different cosmids) that were connected through random end-joining prior to insertion into the nuclear genome (Noutsos et al. 2005). These portions would be too small to detect using only one cosmid probe, but the 19-cosmid mix would hybridize simultaneously to all of the small sequences, potentially allowing detection of the insertion site.

A detailed comparison was performed between B73 and three other inbred lines. The first was Oh43 because this line contained the largest number of mtDNA insertion sites. Five NUMTs detected in B73 were not observed in Oh43 (Figure 2). Oh43 contained at least eight NUMTs that were not observed in B73.

The B73 NUMTs were also compared to those from the A188 inbred line. B73 and A188 differ in their mitochondrial genotypes; A188 carries the rarer NA mitochondrial genotype, of which 5.11% is not represented in the NB mitochondrial genome (Fauron and Casper 1994; Clifton et al. 2004; Allen et al. 2007). A minimum of nine mtDNA insertion sites were detected in B73 that were not identified in A188 (Figure 2). A188 contained five NUMTs that were not detected in B73.

B73 and B37 are the most closely related inbred lines in our study. These two lines were both developed from the Iowa Stiff Stalk Synthetic (BSSS) lines. B37 was selected ∼14 years before B73 (Troyer 1999). Five NUMTs were detected in B73 that were not observed in B37. Fourteen mtDNA insertion sites were identified in B37, with at least one on every chromosome, while no NUMTs were observed on B73 chromosomes 8 and 10 (Figure 2). B37 contained six NUMTs that were not observed in B73.

The strong hybridization signal detected in the B73 9L proximal region (Figure 1b, Figure 2) was seen to be much weaker in B37 (Figure 2). Indeed, B73 was the only line that had such a strong signal from the 19-cosmid mix in this chromosomal region; a lesser signal was observed at this position in seven other lines. KYS had no apparent NUMT at this location. In A188, a NUMT was observed that appeared to be coincident with the centromere of 9. These comparisons suggest that the 9L proximal insertion, containing most of the mitochondrial genome, is not common in maize inbreds and thus is likely a recent event in the B73 lineage.

Analysis of NUMTs within the B73 inbred line:

After observing the extensive diversity of NUMTs among inbred lines we were interested in assaying variation within an inbred line (Figure 3). B73 is a commonly used inbred that is maintained by self and sib pollinations in many laboratories. We obtained the B73 inbred from three different laboratories and have arbitrarily designated the different sources A, B, and C. B73 (A) is the source used for our previous experiments, B73 (B) is from G. Davis (University of Missouri), and B73 (C) is from S. Gabay-Laughnan (University of Illinois). All mtDNA insertion sites indicated previously had been observed in at least 88% of the chromosomes examined. For the purpose of comparing closely related materials, we also included sites that were observed less frequently (63–79% of the examined chromosomes; see supplemental Table 3 at http://www.genetics.org/supplemental/).

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Figure 3.—

Different B73 sources probed with the 19-cosmid mix. The karyotypes of B73 from three different sources are shown. Arrowheads indicate mtDNA insertion sites. White arrowheads indicate insertion sites seen in >88% of the chromosomes observed. Open arrowheads indicate insertion sites seen in 60–87% of the chromosomes observed. Chromosomes were identified as described in Figure 2.

Using the 19-cosmid mix, B73 (A) had 13 detectable NUMTs in at least 88% of the chromosomes examined, and one other site was observed less frequently (on 77% of the chromosomes examined; Table 3 and supplemental Table 3). The insertions were the same as those seen in the earlier analyses (Figure 2). B73 (B) had 13 NUMTs detected in at least 88% of chromosomes examined and 3 less frequently detected sites (Table 3; supplemental Table 3). B73 (C) was more distinctive; 16 NUMTs were observed in at least 88% of chromosomes examined and 2 sites were observed less frequently (Table 3). In particular, a NUMT was observed in the distal region of chromosome 5 that was not observed in B73 (A) and (B). This site could represent a new insertion of mitochondrial sequences that has occurred over the course of a few generations or it could result from the segregation of a preexisting polymorphism.

Most of the mtDNA insertion sites were shared by the various B73 sources (Figure 3). A number of these NUMTs were identified at proximal and distal chromosomal locations (Table 3). In addition, all the B73 sources had equally strong signals on proximal 9L. This relative consistency of detectable NUMTs among different sources of an inbred indicates that the NUMT composition is characteristic for the karyotype of each inbred line. However, the differences within an inbred line illustrate the dynamic nature of NUMT insertions.

A sampling of individual cosmid probes was used to confirm that reproducible differences exist among the mtDNA insertion sites on the chromosomes from different sources of B73 (Figure 4; number of chromosomes examined are given in supplemental Table 4 at http://www.genetics.org/supplemental/).

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Figure 4.—

Different B73 sources (A, B, and C) probed with mtDNA segments. Six individual mtDNA-containing cosmids labeled with Texas red were hybridized to B73 chromosomes of different sources. The white arrowheads mark mtDNA insertion sites and the green arrowheads mark potential cpDNA insertion sites. The eight mix of karyotyping probes was used to identify each chromosome. Only the layer with the Texas red-labeled mitochondrial probes is shown. The marked mtDNA insertion sites were identified on >88% of individual chromosomes examined. Chromosome 8 contained no mtDNA insertion sites and was not included.

Analysis of NUMTs within the W23 inbred line:

The differences observed among the sources of B73 suggest that mtDNA insertions into the nuclear genome of maize can occur frequently. To investigate this phenomenon further, the nuclear chromosomes of two W23 sources were examined for variation in mtDNA insertion sites (Figure 5). Both W23 lineages were originally from the Wisconsin Breeding Program. W23-A (see also Figure 2) was obtained from E. Coe (University of Missouri) and W23-B was obtained from J. Kermicle (University of Wisconsin). Seedlings from each source were characterized for NUMTs and an F₁ hybrid of the A and B sources was generated and analyzed. These karyotypes included mtDNA sites that were observed frequently (at least 88% of chromosomes examined) and those that were more difficult to detect (63–83% of chromosomes examined; supplemental Table 5 at http://www.genetics.org/supplemental/).

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Figure 5.—

Different W23 sources probed with the 19-cosmid mix. The karyotypes of both W23 sources and their F₁ hybrid are included. The top two karyotypes of the separate sources show both the mtDNA insertion sites and karyotyping probes (color). The bottom karyotype of the hybrid shows only the mtDNA insertion sites. Arrowheads indicate mtDNA insertion sites and are labeled as described in Figure 3. The eight mix of karyotyping probes was used to identify each chromosome.

For each source and the hybrid between the two, the numbers and locations of mtDNA insertion sites were examined and compared (Figure 5). W23-A plants had 15 mtDNA insertion sites observed in at least 88% of chromosomes examined and 2 less frequently observed NUMTs (Table 4). W23-B plants had 18 mtDNA insertion sites detected in at least 88% of chromosomes observed and 1 less frequently detected NUMT (Table 4). A majority of the NUMTs within these sources were commonly located proximally and distally.

W23-B had NUMTs located on 1S, 3L, and 6L that were not detected in W23-A. W23-A had only one NUMT (4S) that was not detected in W23-B. The changes occurred in both directions; each source contained at least one mtDNA insertion site that the other source was missing. To confirm the validity of these differences, NUMTs were characterized in the hybrid between the two lines. When a signal was present on a chromosome in one parent and absent in the other, both homologs could be clearly distinguished in the F₁ hybrid (Figure 5). Detecting the differences in the hybrid under identical conditions of tissue fixation and chromosome image capture confirms that these distinctions occur reproducibly. The difference for the mtDNA insertion site on the short arm of chromosome 1 was confirmed with a sampling of individual cosmid probes (Figure 6; numbers of chromosomes examined are given in supplemental Table 6 at http://www.genetics.org/supplemental/). These findings indicate that the NUMT constitution can change within an inbred line.

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Figure 6.—

Two W23 sources (A and B) probed with mtDNA segments. Six individual mtDNA-containing cosmids labeled with Texas red were hybridized to W23 chromosomes of two sources. The white arrowheads mark mtDNA insertion sites. The eight mix of karyotyping probes was used to identify each chromosome. Only the layer with the Texas red-labeled mitochondrial probes is shown. The marked mtDNA insertion sites were identified on >88% of individual chromosomes examined.