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Proc Natl Acad Sci U S A. Jun 29, 2004; 101(26): 9921–9926.
Published online Jun 14, 2004. doi:  10.1073/pnas.0403421101
PMCID: PMC470774
From the Cover
Plant Biology

Dissecting the maize genome by using chromosome addition and radiation hybrid lines

Abstract

We have developed from crosses of oat (Avena sativa L.) and maize (Zea mays L.) 50 fertile lines that are disomic additions of individual maize chromosomes 1-9 and chromosome 10 as a short-arm telosome. The whole chromosome 10 addition is available only in haploid oat background. Most of the maize chromosome disomic addition lines have regular transmission; however, chromosome 5 showed diminished paternal transmission, and chromosome 10 is transmitted to offspring only as a short-arm telosome. To further dissect the maize genome, we irradiated monosomic additions with γ rays and recovered radiation hybrid (RH) lines providing low- to medium-resolution mapping for most of the maize chromosomes. For maize chromosome 1, mapping 45 simple-sequence repeat markers delineated 10 groups of RH plants reflecting different chromosome breaks. The present chromosome 1 RH panel dissects this chromosome into eight physical segments defined by the 10 groups of RH lines. Genomic in situ hybridization revealed the physical size of a distal region, which is represented by six of the eight physical segments, as being ≈20% of the length of the short arm, representing ≈one-third of the genetic chromosome 1 map. The distal ≈20% of the physical length of the long arm of maize chromosome 1 is represented by a single group of RH lines that spans >23% of the total genetic map. These oat-maize RH lines provide valuable tools for physical mapping of the complex highly duplicated maize genome and for unique studies of inter-specific gene interactions.

Plants with one chromosome (monosomic) or one pair of homologous chromosomes (disomic) of an alien donor species added to the entire recipient species chromosome complement serve to dissect the donor genome into individual chromosome entities and separate them from their own genome remnant. The transfer liberates the added chromosome (pair) from the interactive gene expression network of the donor genome and puts the chromosome's genes into the environment of the host genome. This new structural and functional situation can create novel orthologous and nonhomologous gene-to-gene interactions and, hence, helps to answer fundamental questions about gene expression control, inheritance, and syntenic correspondence among different plant species, especially those with large genomes, including maize, with a 1C content of ≈2.7 billion base pairs [Plant DNA C-Values Database (Release 2.0, January 2003), M. D. Bennett and I. J. Leitch, http://rbgkew.org.uk/cval/homepage.html] and a subgenome structure reflecting ancient tetraploidy (1).

By crossing maize to oat, (oat × maize)F1 proembryos were generated, of which 5-10% could be rescued in vitro. Molecular and cytological analyses showed retention of one or more maize chromosomes in addition to the haploid oat genome in 34% of the F1 plants (2-7). Because haploid oat frequently develops unreduced gametes (8), subsequent self-fertilization of (oat × maize)F1 plants with one maize chromosome added to the haploid oat genome (n = 3x + 1 = 22) can produce F2 offspring with one homologous maize chromosome pair added to the doubled haploid (hexaploid) oat genome (2n = 6x + 2 = 44) among other euploid and aneuploid types (9).

A complete series of oat-maize chromosome addition lines (10) has enabled markers and genes to be physically allocated to maize chromosomes without a need for detectable polymorphisms. These unique plant materials confirmed interchromosomal duplicate loci on a large scale, one of the obstacles to whole-genome sequencing. Numerous locus duplications complicate the reassembly of a set of shotgun DNA sequences. Oat-maize chromosome addition lines, however, physically separate these interchromosomal maize orthologs and paralogs from each other and make them accessible to mapping, sequencing, and cloning, even in cases where the duplicated loci of interest carry genes with monomorphic allelic sequences.

A second obstacle that impedes sequencing of the complete maize genome by today's technology is the repetitive nature of ≈85% of the maize DNA. Thus, sequencing strategies are being tested that accomplish the targeted sequencing of less repetitive gene-rich regions (11, 12). These strategies must involve technologies that are capable of arranging those gene islands along the chromosomes and bridging long gaps between contigs. Generating random breaks in the maize chromosome in an identified monosomic oat-maize chromosome addition line and maintaining diminutive maize chromosomes or pieces translocated into oat can provide DNA panels of radiation hybrid (RH) lines, which allow for a presence vs. absence test of markers without the need for polymorphisms (13). With sufficient resolution that is determined by the number and distribution of breaks along the maize chromosomes, RH lines can contribute to placing contigs in the correct order. A panel of RH lines for maize chromosome 9 has demonstrated the efficient mapping of molecular markers (14).

This report summarizes the status for the oat-maize chromosome addition line production and characterization, including the irregular transmission behavior of maize chromosomes 5 and 10 in oat, the last two maize chromosomes recovered as fertile oat-maize addition lines. We also illustrate the development of oat-maize RH lines from addition plants for maize chromosome 1. We show the use of these RH lines for physical mapping and relating genetic map distances to physical chromosome segment sizes.

Materials and Methods

Plant Material. Plants of oat (Avena sativa L.) cultivars GAF-Park, Kanota, Preakness, Starter, Stout, Sun II, and the MN-hybrid (MN97201-1 × MN841801-1) were grown and crossed by maize (Zea mays L.) lines Seneca 60, A188, A619, B73, Mo17, the (A188 × W64A)F1 hybrid, and a line carrying the allele bz1-mum9. More than 200 putative F1-hybrid plants indicating successful (oat × maize) hybridization were recovered and analyzed for vigor, seed set, maize chromosome retention in various tissues, and maize chromosome transmission to F2 offspring (5, 10). F2 plants were tested by PCR-based markers, test crosses, genomic in situ hybridization (GISH), and chromosome counting for presence, stability, and transmission of maize chromosomes added to the oat genome (15). F2 plants were propagated for production of F3 and subsequent generations. Backcross (BC1) plants monosomic for the maize chromosome addition were produced by crossing disomic oat-maize chromosome addition plants back to their corresponding parental oat lines. Batches of 150-300 BC1 seeds were irradiated with γ rays from a 137Cs source at different intensities (20-50 krad) to induce as many maize chromosome breaks as possible without seriously damaging the vigor of the seeds or resulting seedlings. BC1F2 offspring with transmitted maize chromosome deficiencies or oat-maize chromosome translocations (RH plants) were selected by a PCR assay with primers specific for Grande 1 (16) and CentA (17) and used for physically mapping molecular markers to the particular maize chromosome segments.

Genomic DNA Extraction. For a limited number of PCRs (75 or fewer) from single plants, DNA was extracted by the use of the REDExtract-N-Amp Plant PCR kit (Sigma). For larger numbers of PCRs (>75) from single plants, DNA was extracted by using either the DNeasy Plant Mini kit (Qiagen, Valencia, CA) or the acetyltrimethylammonium bromide procedure (18). For labeling and use as probe in GISH experiments, genomic maize DNA was extracted from leaf cell nuclei and purified through a CsCl gradient (19).

PCR. PCRs were accomplished by the use of the REDExtract-N-Amp Plant PCR kit, according to the vendor's recommendations. F1 plantlets and seedlings of the consecutive generations were screened for the presence of maize sequences by using maize-specific primers for the long terminal repeat of the highly dispersed retrotransposon Grande 1 (16) and for the highly centromere-specific retrotransposon-like repeat CentA (17). Individual maize chromosomes or chromosome segments in F1 plantlets, addition lines, and RH seedlings were identified by using maize-specific primers for simple-sequence repeat (SSR) markers (10) that were selected from the maize genetics and genomics database (www.maizegdb.org). BC1F2 plants (putative RH plants) were tested for presence vs. absence of maize chromosome segments by a PCR assay with 45 SSR markers specific for maize chromosome 1 (p-umc1354 to p-umc2244 spanning 1,120 map units, according to the IBM2 map). Markers were selected from the maize genetics and genomics database mentioned above.

Cytology. Root tips (1.5-2 cm) of oat, maize, and oat-maize chromosome addition and RH lines were pretreated, fixed, and stored as described in ref. 10. Root tips for chromosome counting were prepared as described in ref. 20. Meristem cells were squashed in 2% wt/vol Aceto-Orcein (Carolina Biological Supply). Root tips for GISH were prepared as described in ref. 10. Further steps of RNase treatment, postfixation, and in situ hybridization were as described earlier in ref. 21 except that total genomic maize DNA was labeled by the use of the ULYSIS Alexa Fluor 488 nucleic acid labeling kit (Molecular Probes) and probed on slides without using unlabeled competitor DNA. Hybridization was carried out in 40% formamide in 1.5× SSC (225 mM NaCl/22.5 mM trisodium citrate, pH 7.0) at 37°C. Posthybridization stringency washes were carried out in 40% formamide in 1.5× SSC at 42°C. Chromosomes were counter-stained with propidium iodide. Signals were visualized and captured by using an Axioskop microscope equipped for epifluorescence (Zeiss) and a Magnafire charge-coupled device camera (Optronics International, Chelmsford, MA).

Results and Discussion

Maize Chromosome Elimination in (Oat × Maize)F1 Hybrids. In oat × maize crosses, maize chromosomes are occasionally retained (2). This situation is distinct in that the maize genome is completely eliminated in hybridizations between maize and wheat or barley. There is only one report of a maize chromosome being retained in wheat; however, the maize chromosome was not transmitted to offspring (23). The timing of elimination differs as well. The maize chromosome elimination process in oat sometimes extends over longer periods of time compared with that in F1 hybrids generated from wheat × maize and barley × maize crosses (24). In 70% of (wheat × maize)F1 embryos, one or more maize chromosomes were eliminated at the first mitosis. By the eight-cell stage, the embryos had lost all maize chromosomes (24). Maize chromosome elimination from (oat × maize)F1 embryos starts at an early stage in embryogenesis as well (4). However, as an example of the extended time of maize chromosome elimination from oat, in the (oat × maize)F1 plant F1-5133-1 with maize chromosomes 4, 7, and 10 all detected at a young plant stage, a consecutive elimination of individual maize chromosomes was detected by the PCR analysis of genomic DNA extracted from tissues of flag leaves of different tillers from the same plant shortly after meiosis. The first tiller retained only maize chromosome 4, thus eliminated chromosomes 7 and 10. The second tiller eliminated chromosome 10, thus retained chromosomes 4 and 7. The third and fourth tillers eliminated chromosome 7, thus retained chromosome 4 and chromosome 10 as a short-arm telosome (Fig. 1). We observed further instances where maize chromosomes were lost in later growth stages, particularly from plantlets with two or more originally retained maize chromosomes in their complements (results not shown).

Fig. 1.
PCR products from DNA of the F1 (Starter × B73) plant F1-5133-1 when chromosome-specific SSR markers are used; electrophoresis in a 3.5% agarose gel shows the different elimination of maize chromosomes in individual tillers. Lanes 1, young plantlet; ...

(Oat × Maize)F1 Hybrids and F2 Offspring in Different Genetic Backgrounds. Our initial work was conducted with the maize chromosome donor Seneca 60 and the oat recipient Starter. In the last 2 years we have tested several other combinations of maize and oat lines. A total of 201 (oat × maize)F1 plants have been generated from various maize and oat backgrounds, of which 68 F1 hybrids retained one or more maize chromosome(s) in their complements. All 10 maize chromosomes could be recovered, with each occurring at different frequencies as single additions and in combination with other maize chromosomes. No obvious preferential combination for two or more specific maize chromosomes was detected in multiple additions in haploid oats. The frequency of recovery of a particular maize chromosome, the fertility of that plant, and the stability of a maize chromosome appeared to be primarily dependent on the particular maize chromosome interacting with the oat background. Furthermore, the ability to recover a particular maize chromosome in F1 hybrids was not correlated with the ability to produce a fertile addition line for that chromosome. For instance, 20 F1 hybrids with maize chromosome 5 were produced, making the chromosome 5 the most frequently recovered chromosome, either as a single chromosome addition or in combination with other maize chromosomes; chromosome 2 addition plants were the next most frequent with 15 F1 plants produced. Yet, only one of the chromosome 5 plants was fertile and transmitted the chromosome 5 to offspring resulting in the fertile disomic addition OMAd5.59 in Sun II oat background. This finding can be contrasted with chromosome 4, which has been recovered as an addition line nine times, with three of these addition plants being fertile and transmitting the chromosome 4 to offspring. With respect to transmission of maize chromosomes, fertile disomic addition lines fall into different categories. Addition lines carrying chromosomes 2, 3, 4, 6, and 9 exhibited little or no problems with transmission (nearly 100% maternal and paternal transmission rate). Lines carrying chromosomes 1 and 7 initially had poor transmission of the maize chromosome, but after several generations they show good transmission of the maize chromosome (>80% transmission rate), possibly due to selective breeding for stable diploid offspring. Chromosomes 1, 5, and 8 additions have fertility problems even after several generations of selection, and chromosome 10 additions have transmitted only short-arm derivatives to offspring. Eleven disomic additions for different maize chromosomes, including 2, 4, 5, 6, 7, 8, and 9 in different oat backgrounds, have been added to the previously reported set (15), making a total of 50 fertile addition lines (Table 1).

Table 1.
Fertile oat-maize chromosome addition lines

Irregular Maize Chromosome 5 Transmission in Oat. Disomic maize chromosome 5 additions recovered earlier in two different oat backgrounds (OMAd5.09 in Starter and OMAd5.17 in MN-hybrid) show significantly diminished paternal transmission, whereas maternal transmission of the maize chromosome 5 is only moderately reduced. Crossing a OMAd5.09 plant as male back to Starter produced four monosomic maize additions among 58 BC1 offspring, giving a paternal transmission rate of 6.9% (4 of 58). In 30 F4 offspring of a disomic addition from the line OMAd5.09, only 2 plants were disomic additions, which corresponds to a minimal paternal transmission of 6.7% (2 of 30). Twenty-one plants were monosomic additions, which indicates a probable maternal transmission of 76.7% (23 of 30), although one or two of the monosomic additions could be from paternal transmission. In a further experiment analyzing 25 F4 offspring of a disomic addition from the line OMAd5.17, only two plants were disomic additions, indicating a paternal transmission of 8% (2 of 25). Seventeen plants were monosomic additions, which indicates a probable maternal transmission of 76% (19 of 25). Taking data for both oat backgrounds together, the irregular low paternal transmission of the maize chromosome 5 (6.7-8%) clearly accounts for the low frequency of disomic addition offspring from disomic chromosome 5 addition plants.

Maize Chromosome 10 Transmission. Chromosome 10 is the smallest chromosome, with 190 Mbp in the Seneca 60 complement. Yet, it was the most frequently eliminated chromosome in (oat × maize)F1 hybrids, indicating a low tolerance for its presence in oat (4, 10). The first recovered monosomic addition of chromosome 10 occurred in a haploid of GAF-Park oat background (10). Since that time, the plant has been vegetatively propagated by tiller cloning under short-day conditions. Periodically, tiller clones have been moved to a long-day regime to induce flowering for self-pollination or backcrossing with GAF-Park pollen. After screening thousands of spikelets over a period of ≈3 years, we recently recovered one seed. This one seed, however, did not possess maize chromatin; thus, a maize chromosome addition offspring has not been produced by this plant.

In this new series of oat × maize crosses, we recovered 11 plantlets that retained maize chromosome 10 as single or multiple chromosome additions, 9 plantlets in Starter oat background, and 2 plantlets in Sun II background. One chromosome 10-positive F1 plant (F1-0289-1, Seneca 60 × Sun II) set 189 F2 seeds in its first four panicles (Table 2). The PCR assay with Grande 1 showed that 28 F2 plants originating from three panicles were maize-positive, and 2 F2 plants were maize-negative. GISH analyses revealed 16 disomic and 12 monosomic addition plants. All 10 F2 plants originating from the fourth panicle were maize-negative. In all 28 maize-positive plants, only chromosome 10 short-arm-specific SSR markers (p-phi041, pphi117, and p-umc1293) were present; none of the long-arm-specific markers (p-umc1249, p-umc1196, p-umc1176, and pumc1084) tested was detected (22). We, therefore, assume that the chromosome transmitted to offspring in every case is a short-arm telocentric derivative of chromosome 10 (22). The derived line disomic for the chromosome 10 short-arm telocentric was labeled OMAdt10S.20 and seeds (F3 offspring) have already been distributed (Table 1).

Table 2.
Seed set from fertile oat-maize chromosome 10S additions

The F1 hybrid F1-5133-1 originated from crosses of Starter oat with maize B73. This hybrid possessed the three maize chromosomes 4, 7, and 10 in addition to the haploid oat complement at a young growth stage. In DNA samples from both the third and fourth tillers (F1-5133-1/c and F1-5133-1/d), SSR markers were present for chromosome 4 and chromosome 10. Although all three short-arm-specific markers for chromosome 10 were present in the two DNA samples, neither sample showed evidence for long-arm-specific markers. Therefore, we assume that chromosome 10, which was accompanied by maize chromosome 4, also was a telocentric short-arm derivative of chromosome 10. PCR analysis of the three F2 offspring from the panicles of the third and fourth tiller of plant F1-5133-1 (Table 2) showed that only two F2 plants had the short-arm-specific SSR markers for chromosome 10, and none had any of the long-arm-specific SSR markers for chromosome 10. All three F2 plants had the chromosome 4-specific SSR markers. All six F2 offspring from the F1-5133-1/a panicle were positive for B73 chromosome 4, three as monosomic and three as disomic additions. The tiller F1-5133-1/b did not set seed.

The generation of a fertile disomic telocentric addition for chromosome 10 (OMAdt10S.20) is a major breakthrough in our efforts to develop a complete series of fertile oat-maize addition lines (22). However, this observation raises the question of why does only the short arm of an added maize chromosome 10 transmit in oat. Does the long arm possess a gene that prevents transmission in this alien background? High sterility occurs in the highly stable whole chromosome 10 addition in GAF-Park oat and the two independent events of short-arm derivatives of chromosome 10 in Sun II and Starter oat, where the long-arm telocentrics could not be established. The situation appears similar to the difficulties of generating a disomic euplasmic addition line for Betzes barley chromosome 1H and for its long-arm telosome 1HL in Chinese Spring wheat (25). The difficulties in wheat (26-28) appear to be caused by the interaction of the gene Shw (sterility in hybrid with wheat) with the wheat background causing sterility. However, the sterility was alleviated by the simultaneous addition of monosomic or disomic chromosome 6H to the 1H addition (29). Perhaps we could select (oat × maize)F1 hybrids for the simultaneous additions of other chromosomes with chromosome 10 to possibly allow fertility and transmission of the whole chromosome 10. In addition, it may be feasible to use additional maize genotypes that possess a different allele of the presumed gene on chromosome arm 10L responsible for the sterility. In the corresponding wheat-barley addition situation, chromosome 1H of the closely related wild barley (Hordeum vulgare L. subsp. spontaneum) was added to wheat without causing a severe effect of sterility (30).

Oat-Maize Chromosome 1 RHs. Monosomic oat-maize chromosome 1 addition seeds, the foundation for the development of oat-maize chromosome 1 RH lines, were treated with γ rays at two levels. These levels were 180 BC1 seeds treated with 40 krad and 120 BC1 seeds treated with 35 krad. A total of 46 maize-positive plants, as indicated by the presence of the markers Grande 1 and/or CentA, were recovered from the 40-krad treatments. The 35-krad treatment generated 54 maize-positive plants. These 100 BC1 plants were allowed to self-pollinate. Of these, 91 panicles produced 340 BC1F2 offspring that tested negative and 171 BC1F2 offspring that tested positive for maize chromatin in their genomes, indicating successful transmission of maize segments. It is notable that after the γ radiation treatment of 300 monosomic addition seeds, only 100 plants retained their maize chromosomes or a diminutive maize chromosome derivative. This finding indicates that a majority of the breaks generated maize fragments that were eliminated from somatic tissues. Earlier results showed a certain level of somatic instability for whole chromosome 1 addition plants resulting in chromosome loss (7, 10).

A set of 45 SSR markers distributed along maize chromosome 1 was used to determine by a presence vs. absence test for each marker approximate points of maize chromosome breakage in the 171 BC1F2 plants. All 45 SSR markers were present in 98 BC1F2 plants, which represent 50 families. These plants were considered as possessing either a whole maize chromosome without a break or a reciprocal oat-maize translocation. These plants will be self-pollinated, and offspring of those with reciprocal translocations will be selected for the segregating translocated chromosomes. Nine BC1F2 plants, forming five families, showed complex rearrangements, including interstitial deletions and multiple translocations with oat. Forty-four BC1F2 plants constituted 21 families, each representing one likely independent (chromosome rearrangement) event (Table 3). These 44 RH plants were placed into 10 panel groups, with plants within a group resulting from similar maize chromosome breaks based on marker analysis (Table 3 and Fig. 2). Fig. 2 illustrates the definition of eight segments by seven breaks in selected RH lines for maize chromosome 1 representing the 10 groups (Table 3). Plants with only one break in their maize chromosome, and thus possessing only one deficiency or one oat-maize translocation, are shown in the first panel (Fig. 2). The markers shown in the left column are the first and last marker present or absent and frame the breakpoints. The points define six segments on the short arm (p-umc1354 to p-umc168), one large segment spanning the centromere region (p-umc1626 to p-mmc0041), and one additional segment on the long arm (p-bnlg1720 to p-umc2244).

Fig. 2.
Panel of the first RH lines for maize chromosome 1. Shown are the 15 SSR markers that frame the seven breakpoints, hence define the RH segments between p-umc1354 (most distal on the short arm) and p-umc2244 (most distal on the long arm) markers representing ...
Table 3.
Groups of RH lines from independent chromosome mutation events that define the same breakpoints on chromosome 1

The apparently single breakpoint in the long arm of chromosome 1 as defined by groups 9 and 10 is represented by seven independent breakage events. This break is marked proximally by the SSR marker p-mmc0041 and distally by the SSR marker p-bnlg1720 (Fig. 2). The portion (7 of 21) of lines that break at the same point or very similar points indicates a preferential breakage and/or transmission of the chromosome segments to offspring. On the short arm, five independent events (5 of 21) demonstrate a common break in the range that is marked by the SSRs p-umc1071 and p-umc1727 defining segment 1. All other breakpoints are defined by one or two events. Most striking is that we did not observe a centric break resulting in either a telocentric maize chromosome or a centric maize-oat translocation. This situation left intact a large area spanning the centromere and the proximal regions on both arms, segment 7 (Fig. 2), and it is in strong contrast to earlier results from the production of maize chromosome 9 RHs (12) that showed that the level of chromosome 9 breakage across the chromosome was relatively constant.

The physical sizes of the segments differ remarkably as shown by GISH experiments (Fig. 3). In the line 1.07.3-001.3-03 (a sibling from group 7), the maize chromosome shows a primary constriction that defines the deficient short arm to ≈80% of its regular WT metaphase length. This result would mean that the missing element (20%) represents a genetic size of at least 445 map units separated into 6 distinct segments by 8 of 10 groups. On the other hand, GISH of line 1.07.2-007.3-04 (a sibling from group 9) shows the distal maize chromosome fragment translocated to an oat chromosome. The fragment length corresponds to ≈20% of the long-arm WT length in metaphase and visualizes segment 8. Even considering that the definition of the single segment by two markers varies over a considerable genetic distance, the segment 7 spans approximately the proximal 80% of the short and the proximal 80% of the long arm of the genetic map of maize chromosome 1. The line 1.07.1-020.3-01 (sibling of group 3) shows by GISH analysis a fragment of ≈15% of the WT short-arm length translocated to an oat chromosome. This fragment visualizes the length of the segments 1 and 2 together marked by SSRs p-umc1397 and p-umc1479.

Fig. 3.
GISH of metaphase chromosomes from root tips of three RH plants of the maize chromosome 1 panel. (A) Plant BC1F2, 1.07.3-001.3-3 (sibling of group 7), arrow points to the deficient short arm of maize chromosome 1; the chromosome lost ≈20% of its ...

Summary

The current set of disomic oat-maize addition lines involves all maize chromosomes in different oat backgrounds with the exception of chromosome 10. The maize chromosome 10 addition progeny has only the short arm; a fertile disomic telocentric addition line is available. The whole chromosome 10 added to haploid GAF-Park oat does allow the availability of DNA. Although not fertile, and therefore not capable of producing disomic addition offspring, we continue to maintain the original plant vegetatively by tiller cloning under short-day growing conditions. The leaves show remarkable somatic stability for the added maize chromosome over a period of >3 years. The plant serves as a source for chromosome 10 genomic DNA and RNA.

The complete series of DNAs made from each maize chromosome addition has been used as a powerful tool to allocate genes and markers to chromosome. Ananiev et al. (31) used the oat-maize chromosome 9 addition line as the DNA source to construct a chromosome-specific cosmid library allowing the isolation of maize-specific repetitive DNA families. The low level of cross-hybridization under standard conditions between oat and maize genomic DNA makes it possible to screen libraries for maize species-specific sequences (31).

Oat-maize addition lines are ideal for mapping gene families and markers that have more than one copy on different chromosomes likely because of the duplicative nature of maize. For example, Okagaki et al. (32) mapped 350 ESTs and sequence tagged sites to chromosomes by a presence vs. absence test and demonstrated the usefulness of the complete addition line set. However, the true power of the addition lines as a tool for maize genomics and genetics may be that no marker polymorphism is required for large-scale mapping. The value of the plant material for gene expression studies has already been shown in an analysis of interchromosomal interaction with respect to expression of the maize gene liguleless 3 on chromosome 3 (33) or the reduced susceptibility against the fungal pathogen Puccinia coronata f. sp. avenae in the oat-maize chromosome 5 addition lines (unpublished data).

With the development of RH lines from the oat-maize additions, markers can be placed to chromosome regions. Visualization by GISH of the rearranged maize chromosome fragments together with marker data helps to relate physical sizes to genetic distances along the chromosome arms. Besides the use of addition and radiation hybrid lines for mapping purposes, the extensive dissection of the maize genome provides powerful material for the targeted cloning of chromosome-specific DNA and to study chromosome-specific structures and their behavior in an alien background.

Acknowledgments

This is a joint contribution of the Minnesota Agricultural Experiment Station and the Department of Agriculture-Agricultural Research Service. This material is based upon work supported by the National Science Foundation Grant 011134.

Notes

This report was presented at the International Congress “In the Wake of the Double Helix: From the Green Revolution to the Gene Revolution,” held May 27-31, 2003, at the University of Bologna, Bologna, Italy. The scientific organizers were Roberto Tuberosa, University of Bologna, Bologna, Italy; Ronald L. Phillips, University of Minnesota, St. Paul, MN; and Mike Gale, John Innes Center, Norwich, United Kingdom. The Congress web site (www.doublehelix.too.it) reports the list of sponsors and the abstracts.

Abbreviations: BC, backcross; GISH, genomic in situ hybridization; RH, radiation hybrid; SSR marker, simple-sequence repeat marker.

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