Mitochondrial DNA transfer to the nucleus generates extensive insertion site variation in maize

Abstract

Mitochondrial DNA (mtDNA) insertions into nuclear chromosomes have been documented in a number of eukaryotes. We used fluorescence in situ hybridization (FISH) to examine the variation of mtDNA insertions in maize. Twenty overlapping cosmids, representing the 570-kb maize mitochondrial genome, were individually labeled and hybridized to root tip metaphase chromosomes from the B73 inbred line. A minimum of 15 mtDNA insertion sites on nine chromosomes were detectable using this method. One site near the centromere on chromosome arm 9L was identified by a majority of the cosmids. To examine variation in nuclear mitochondrial DNA sequences (NUMTs), a mixture of labeled cosmids was applied to chromosome spreads of ten diverse inbred lines: A188, A632, B37, B73, BMS, KYS, Mo17, Oh43, W22, and W23. The number of detectable NUMTs varied dramatically among the lines. None of the tested inbred lines other than B73 showed the strong hybridization signal on 9L, suggesting that there is a recent mtDNA insertion at this site in B73. Different sources of B73 and W23 were examined for NUMT variation within inbred lines. Differences were detectable, suggesting either that mtDNA is being incorporated or lost from the maize nuclear genome continuously. The results indicate that mtDNA insertions represent a major source of nuclear chromosomal variation.

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.

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.

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.

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.

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.

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.

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.