Maize chromosome additions and radiation hybridsin oat and their use in dissecting the maize genome

HOWARD W. RINES1,2,*, RONALD L. PHILLIPS1, RALF G. KYNAST1,RON J. OKAGAKI1, WADE E. ODLAND1, ADRIAN O. STEC1,

MORRISON S. JACOBS1 AND SHANNON R. GRANATH1

1Department of Agronomy and Plant Genetics, and Center for Microbial andPlant Genomics, University of Minnesota,

411 Borlaug Hall, 1991 Upper Buford Circle, St. Paul, MN 55108, USA2Plant Science Research Unit, U.S. Department of Agriculture-Agricultural Research Service,

411 Borlaug Hall, 1991 Upper Buford Circle, St. Paul, MN 55108, USA

*Corresponding author: rines001@umn.edu

Abstract

Plant recovery from wide-cross sexual hybridization between members of two sub-families of grasses, as initially described in the production of haploid wheat plantsin wheat ✕ maize crosses, can also be obtained in oat ✕ maize crosses. In wheat ✕

maize crosses, haploid wheat plants are obtained as a result of complete eliminationof the maize chromosomes prior to embryo rescue. In contrast, about one-third ofthe plants from oat ✕ maize crosses contain one or more maize chromosomes inaddition to a complete haploid complement of 21 oat chromosomes. Over the pastfew years, all ten maize chromosomes have been recovered as single chromosomeadditions to a haploid oat complement, and fertile disomic addition lines in oathave been obtained for maize chromosomes 1 through 9 as well as a ditelosomicfrom maize chromosome 10 short arm. Polymerase chain reaction (PCR) assays ofDNA extracts for the maize-specific Grande-1 dispersed repeated element are usedto detect plants with maize chromatin. Assays with maize chromosome-specificmarkers are used to identify which maize chromosome(s) is present, and genomicin situ hybridization analysis of root-tip cells is performed to visualize the numberof maize chromosomes present. Plant phenotypes, transmission characteristics ofaddition chromosomes, and potential uses of the oat-maize addition lines aredescribed here. We also emphasize the value of these strains for mapping dupli-cated maize sequences and gene families. Gamma irradiation of oat-maize additionlines is used to produce radiation hybrid lines with maize chromosomal segmentdeletions and translocations. Finally, we describe the use of radiation hybrids

Tuberosa R., Phillips R.L., Gale M. (eds.), Proceedings of the International Congress “In the Wake of the Double Helix: From the Green Revolution to the Gene Revolution”,27-31 May 2003, Bologna, Italy, 427-441, ©2005 Avenue media, Bologna, Italy.

Rines-4 26-10-2005 9:24 Pagina 427

428

having overlapping deficiencies to enable physical mapping of maize sequences tomaize chromosome segments.

Introduction

Self-fertile partial hybrids between oat and maize can be formed from sexual crossesbetween the two species. Not many years ago most people would have consideredit improbable that such a hybrid combination could be produced between two suchdistantly related cereals. Partial and complete hybrids between species within thesame genus and even between species of different genera within the same tribe ofcereals, such as among wheat, barley, rye and various Triticeae wild relatives, hadbeen known for over a century (Sharma and Gill 1983). These related species wereconsidered secondary or tertiary gene pools to be tapped for genes controllingdesired traits, such as pathogen resistance. Expectations of wider species combina-tions from somatic cell hybridization arose in the 1970s when interspecies somaticcell combinations were first produced by protoplast fusion (Carlson et al. 1972).Wide interspecific somatic combinations such as ones involving wheat and maizehave been described. In a recent report, random amplified polymorphic DNA(RAPD) marker and genomic in situ hybridization (GISH) analysis of plantsregenerated from wheat-maize somatic hybrids indicated small islands of wheatchromatin incorporation into sterile plants that resembled the maize parent butwith no recognizable wheat morphological traits (Szarka et al. 2002).

A significant breakthrough in the sexual wide-cross barrier in the grassesoccurred with a 1986 report by Laurie and Bennett. They presented cytological evi-dence of hybrid zygote formation in a cross spanning two subfamilies of theGramineae, the Pooideae and the Panicoideae, specifically wheat (Triticum aestivumL., 2n = 6x = 42) by maize (Zea mays L., 2n = 2x = 20). This work was a follow-upto a report by Zenkteler and Nitsche (1984) of cytological observations ofearly-stage embryo formation in wide-cross combinations. In their report, Laurieand Bennett (1986) observed that although zygotes were formed with 21 wheatchromosomes plus 10 maize chromosomes, the chromosome complement of thedeveloping embryo cells was rapidly reduced to a haploid set of 21 wheat chromo-somes by uniparental genome loss. The loss of maize chromosomes in the wheat bymaize combination occurs within the initial three cell divisions (Laurie and Bennett1989). Several reports by these and other workers followed, revealing thatwide-cross combinations between various species of Triticeae as the female and awide variety of Panicoideae species including sorghum, pearl millet, teosinte, Trip-sacum and Coix as male showed a similar pattern of hybrid zygote formation withuniparental loss of the donor pollen chromosomes during subsequent embryodevelopment (e.g. Laurie and Bennett 1988a; Laurie et al. 1990; Ushiyama et al.1991; Riera-Lizarazu and Mujeeb-Kazi 1993; Mochida and Tsujimoto 2001).

RINES ET AL.

Rines-4 26-10-2005 9:24 Pagina 428

429

An important aspect stimulating the study of wheat ✕ maize and similar crosses wasthe finding that post-pollination treatments with plant growth substances enhancedcaryopsis and embryo development, even in the absence of endosperm develop-ment, such that embryos with a complete haploid set of maternal wheat chromo-somes could be rescued on culture medium (Laurie and Bennett 1988b; Suenagaand Nakajima 1989; Riera-Lizarazu and Mujeeb-Kazi 1990). These treatments usual-ly included synthetic auxins such as 2,4-dichlorophenoxyacetic acid administeredeither through direct application onto florets 24-48 hours after pollination or byinjection or culm culture uptake into the maternal plants. Subsequent germinationof the rescued embryos gave haploid wheat plants that with colchicine treatment orother chromosome doubling techniques could produce doubled haploids forbreeding and genetic purposes. This approach for haploid production as an alter-native to anther/microspore culture technologies has the important feature of beingless genotype dependent. The wide-cross approach involving crosses of Poideaespecies by Panicoideae species, usually wheat by maize, with subsequent uniparentalgenome loss has been adopted for use in bread (T. aestivum) and durum (T.turgidum) wheat breeding programs, development of doubled haploid mappingpopulations and production of wheat alien disomic addition and substitution linesas key intermediates for alien gene introgression (Mujeeb-Kazi and Riera-Lizarazu1996).

Oat by maize crosses

Early attempts using anther culture to produce haploids and doubled haploids inoat (A. sativa L.) for breeding and genetic studies were of limited success withonly three plants recovered from more than 80,000 anthers plated (Rines 1983).Based on the reports of haploid wheat plants recovered from wheat by maizecrosses, we initiated efforts to produce oat haploids by crossing oat by maize. Oathaploid plants were recovered, although at a lower frequency per floret pollina-tion than was obtained for wheat haploids from wheat by maize crosses (Rinesand Dahleen 1990). One novel aspect of the oat haploid plants noted in contrastto haploid wheat plants was that the haploid oat plants were often partiallyself-fertile as a result of meiotic restitution yielding unreduced gametes. Thus, oatdoubled haploids could be recovered without colchicine treatment. Another dif-ference soon observed in the oat ✕ maize crosses was that some of the recoveredplants retained one or more maize chromosomes (Rines et al. 1995;Riera-Lizarazu et al. 1996). In fact, among more than 400 plants recovered in oat✕ maize crosses made through the years, one-third have one or more maize chro-mosome(s) present as alien additions to a complete haploid complement of 21oat chromosomes (Kynast et al. 2004).

Furthermore, many plants with only one and on rare occasion those with more

RINES ET AL.

Rines-4 26-10-2005 9:24 Pagina 429

retained maize chromosomes are partially self-fertile and yield doubled haploid pro-genies disomic for one (2n = 6x +2 = 44) or rarely two maize chromosomes. In con-trast, thousands of wheat haploids have been produced from wheat ✕ maize crosseswith only one report of a maize chromosome retained and it was not transmittedto progeny (Comeau et al. 1992). Thus, oat appears distinct from wheat in itsapparent tolerance and propagation through generations of added alien maize chro-mosomes. The basis or mechanism of this disparate oat-maize chromosomal toleranceinteraction is not known although Jin et al. (2004) have reported that centromeresof added maize chromosomes in oat plants make use of oat centromeric histoneCenH3. This situation is true even in a maize chromosome 6 addition which hasthe maize CenH3 gene present, but the maize gene is apparently silenced (Jin etal. 2004).

Fertile maize chromosome addition plants are recovered at a low frequency ofonly about 0.1% of oat florets that are maize pollinated. To date, about 80 maizechromosome addition plants have been recovered and characterized (Kynast et al.2004). Most of these involve a chromosome of maize cv. Seneca 60, a sweetcornhybrid, added to oat cv. Starter. However, through the use of additional varieties ofoat, particularly Sun II, all ten maize chromosomes have been recovered as singleadditions to a haploid oat complement. As a result of meiotic restitution in thesehaploid plants, self-fertile disomic additions (2n = 6x + 2) have been recovered formaize chromosomes 1 through 9 and also a ditelocentric addition for the short armof maize chromosome 10. The occurrence and transmission of only the short armof chromosome 10 has been observed in three independent partial hybrids (Kynastet al. 2004). This situation may be analogous to that observed in barley chromo-some additions to wheat where only the short arm of barley chromosome 1H wastransmitted, apparently due to the presence of a sterility-causing gene (Shw) on thelong arm of 1H when it is present in wheat (Taketa et al. 2002).

The detection of maize chromatin in an oat background is made by a PCR assayfor the presence of Grande-1 elements (Kynast et al. 2001), which are highlyrepeated retrotransposon elements distributed throughout the maize genome(Monfort et al. 1995; Meyers et al. 2001) but are absent in oat (Fig. 1). The iden-tity of the maize chromosome or chromosomes in Grande-1 positive plants is thenrevealed using a set of maize-chromosome-specific simple sequence repeat (SSR)markers selected from the maize genetic map (www.maizegdb.org) as described inKynast et al. (2001) (Figure 2). Determination of the number of maize chromo-somes present in an oat-maize addition plant is made by GISH analysis of root-tipcells with fluorochrome-labeled maize genomic DNA as probe (Kynast et al. 2001,2002). Such chromosome number determinations are particularly important inprogeny lines of single maize chromosome additions to reveal if the maize chromo-some is present in a disomic or monosomic state.

RINES ET AL.

430

Rines-4 26-10-2005 9:24 Pagina 430

431

RINES ET AL.

Figure 1. Identification of oat ✕ maize cross derivatives that have a maize chromosome(s) presentusing PCR analysis for a maize-specific repeat, Grande-1.

Figure 2. Identification of maize chromosomes 4 and 5 as retained maize chromosomes in an F1(oat ✕ maize) plant by PCR analysis using sets of SSR primers for markers specific to each of the tenmaize chromosomes.

Mar

ker

Oat

Mai

zeF1 (Oat ✕ Maize) plants+ + +

M 1 2 3 4 5 6 7 8 9 10

Rines-4 26-10-2005 9:24 Pagina 431

Features of oat-maize chromosome addition lines

Although each of the ten maize chromosomes has been recovered as an individualaddition to an oat genome, they differ in frequency of recovery, stability of mitoticand meiotic transmission and phenotypic effects on the plants. Furthermore, theseaspects can also be influenced by the genotypes of both the oat genome host and themaize chromosome donor. For example, chromosome 5 has been the most frequent-ly retained maize chromosome in oat ✕ maize F1 plants having been recovered inapproximately 70% of the F1 plants with retained maize chromosomes; however, inonly three of these has this maize chromosome been transmitted to F2 progeny fol-lowing self-fertilization (Kynast et al. 2004). In the cases where Starter-1 is the hostoat genome, the Seneca 60 maize chromosome 5 has proven to be somewhat somati-cally unstable requiring screening of tillers to determine if it has been retained and inprogenies if it is present in a disomic, monosomic, or even intact state. Other chro-mosome additions including ones for maize chromosomes 2, 3, 4, 6 and 9, while lessfrequently recovered initially in oat ✕ maize F1s, behave in a much more stablefashion with nearly 100% transmission frequencies as disomics through generations.Maize addition chromosomes 1 and 7 were transmitted in an irregular fashion in ini-tial generations after recovery; however, with selection for higher transmitting proge-ny, these two chromosome additions have become more regular in their transmissionin later generations. Chromosome 8 additions when present in a disomic state havesuch reduced vigor and fertility that chromosome 8 is maintained only as a mono-somic addition line. The chromosome 8 and other monosomic additions of maizechromosomes in oat exhibit meiotic transmission behavior similar to that shown inother types of interspecific monosomic alien additions with 5-15% transmissionthrough the female gametophyte and none through the male, the latter likely due topollen competition. Meiotic transmission for maize chromosome 10 additions hasbeen observed only for the short arm. However, an addition plant for an intact maizechromosome 10 has been maintained through clonal propagation of a primary oat ✕maize F1 haploid, thus providing a ready source of DNA of this addition.

The presence of an added maize chromosome in these oat-maize addition linesoften leads to morphological and other phenotypic aberrations, the nature of whichdepends on the particular maize chromosome present and somewhat on the genotypeof the oat host. These phenotypes have been described in detail by Kynast et al.(2001). Some of the more striking phenotypes include a hooked panicle and abnor-mal ligules in the upper leaf blade attachment sites of a chromosome 3 addition in anoat Sun II background. Expression of the maize Liguleless 3 (Lg3) gene was demon-strated by RNA expression analysis in the ligule region of maize chromosome 3 addi-tion Sun II plants (Muehlbauer et al. 2000). The ectopic expression in these plants ofthe maize Lg3 gene located on maize chromosome 3 is probably a result of its sepa-ration from its presumed regulating factor produced by the rough sheath (rs2) gene onmaize chromosome 1. Other notable phenotypes that have been observed include

432

RINES ET AL.

Rines-4 26-10-2005 9:24 Pagina 432

thickened stems and compact panicles in maize chromosome 1 addition plants, uppernodal branching in chromosome 5 additions, disease mimic lesions in older plants ofchromosome 6 additions and erratic rapid onset plant necrosis in chromosome 9additions (Kynast et al. 2001).

Various observations indicate that some genes on addition maize chromosomesmay be silenced in an oat genomic background. For example, a lack of an additionalnucleolus associated with a maize chromosome 6 addition indicates that the NORregion on that maize chromosome is silent in an oat background. A light-sensitive redanthocyanin pigmentation pattern was observed in a primary oat-maize F1 plant witha maize chromosome 2 present and could be explained by expression of the B1(booster1) gene present on chromosome 2 of the maize donor, but this coloration wasnot observed in subsequent generations of this chromosome 2 addition line.

Utility of oat-maize chromosome addition lines

The expression of added maize genes in a primarily oat genetic background inoat-maize addition lines provides excellent opportunities for analyzing regulation andphenotypic effects in interspecific situations. These materials not only allow separa-tion of genes on one chromosome of maize from interactions with those on othermaize chromosomes, but also may serve to add useful new traits to oat. For example,Kowles and associates (2004, personal communication) have recently obtained evi-dence of C4 photosynthetic component expression in oat-maize chromosome addi-tion lines. A C4-specific phosphoenolpyruvate carboxylase protein and enzymaticactivity was detected in a maize chromosome 9 addition and C4–type cell structuresurrounding vascular tissue with increased chlorophyll content was observed histo-logically in a chromosome 3 addition to oat, the C3 species. Also, a reduction in su-sceptibility to the oat crown rust pathogen Puccinia coronata avenae has been observedin oat lines with an added maize chromosome 5 (unpublished results).

The oat-maize addition lines have been used in several other studies includinganalysis of meiotic pairing behavior of a single maize chromosome pair, where themaize chromosome pair can be distinctly visualized from the background of oat chro-mosomes by “painting” them with fluorochrome-labeled maize genomic DNA probe(Bass et al. 2000), and in cytometric flow-sorting of a single maize chromosome (Liet al. 2001). However, their most significant use has been as tools for physical map-ping of maize DNA sequences. By use of a series or panel of DNAs extracted fromthe ten individual maize chromosome addition lines in PCR or Southern hybridiza-tion assays, one can assign maize sequences to chromosomes by a presence/absenceassay without a need for polymorphism. Such assignments are particularly valuablefor sorting out loci for members of gene families as illustrated in Figure 3. Additionline use in assignment of genes to chromosome is similar to early work using humanchromosome additions in human-rodent somatic cell lines where human chromo-

433

RINES ET AL.

Rines-4 26-10-2005 9:24 Pagina 433

somes were reduced in number due to uniparental chromosome elimination fromwhat were initiated as human-rodent somatic cell hybrids (Ruddle 1973). Such humanchromosome addition lines in somatic cells were initially used to assign specific pro-tein-encoding genes to chromosome and then became important for direct DNAsequence assignment to chromosome prior to the development of more specificphysical mapping of sequences to human chromosome region using radiation hybrids.

The ability to sort out duplicate loci or closely related sequences by assignment todifferent chromosomes may prove to be a much greater necessity in trying to assem-ble a complete sequence for the maize genome than it was for the human genomebecause of an estimated 60-82% of the maize genome composed of duplicated seg-ments (Gaut 2001) versus only 5-6% of the human genome occurring as segmen-tal duplications (Bailey et al. 2002). The ancient tetraploid origin of maize greatlyincreases the genome complexity to be sorted out due to the presence of orthologousloci tracing to different ancestral donor species as well as paralogous loci originatingfrom within the species genome such as by tandem duplication. Use of theseoat-maize addition lines to determine the chromosome location of duplicated orfamilies of gene sequences was demonstrated in a study by Okagaki et al. (2001) inwhich more than 350 previously unmapped expressed sequence tag (EST) andsequenced-tagged site (STS) sequences were mapped to maize chromosomes.Analysis of the mapping patterns of duplicated or homologous loci provide insightsinto the duplicated genomic structure and orthologous chromosomal segment rela-tionships in modern maize and its putative ancient allotetraploid origin.

434

RINES ET AL.

Figure 3. Mapping to maize chromosome members of a gene family using PCR assays of DNAsfrom a set of lines each with one of the ten maize chromosomes present as a single addition in oat.Electrophoresis of products from PCR using primers for a Zmet1 sequence showed only a singleband with maize lines B73 and Seneca 60 but revealed family members present on maize chromo-somes 5, 7 and 10.

Rines-4 26-10-2005 9:24 Pagina 434

Oat-maize radiation hybrids

The parceling of the ten chromosomes of maize into individual chromosomeadditions of oat serves to reduce the complexity of the maize genome about10-fold. To reduce the complexity even more by creating oat lines with only asegment(s) of an individual maize chromosome present, we treated single maizechromosome addition seed with gamma radiation to break up the maize chro-mosome. We then selected among progeny for offspring each with only a por-tion(s) of the maize chromosome present. Even though the oat chromosomes alsoare affected by the radiation treatment, the allohexaploid nature of the oatgenome provides a high level of buffering and tolerance to chromosomal rear-rangements. As material to be irradiated, disomic maize chromosome additionlines are first crossed back to their oat parent to produce monosomic maize addi-tion seed with only a single copy of the maize chromosome; thus induced lossesof portions of the maize chromosome can be more readily detected and inter-preted. Induced maize chromosome segment losses are detected in progeny of theplants grown from irradiated seed and thus are meiotically transmissible. Cyto-logical and molecular marker analysis of materials recovered from the first maizechromosome addition line so treated, a maize chromosome 9 addition line,revealed a variety of maize chromosome segmental losses and rearrangements(Riera-Lizarazu et al. 2000). These included both maize chromosomes with deletedsegments and oat-maize reciprocal and non-reciprocal translocations. Cytologicalobservations by GISH visualized the types of rearrangements. The extent ofmaize chromosome 9 presence/loss in these lines, which are termed radiationhybrids, was characterized by assays with a series of maize markers that are dis-tributed along the maize genetic map. Assemblage of a series of these radiationhybrids with overlapping segments allows assignment of a maize sequence previous-ly assigned to chromosome 9 to be located to a particular region defined by tworadiation hybrid breakpoints. The initial set of chromosome 9 radiation hybridscontained 27 segments defined by 26 breakpoints. Thus, if the breakpoints wererandomly distributed along maize chromosome 9, which has an estimated size of~200 Mbp based on the chromosome 9 length fraction of the total maize genomeof ~2,500 Mbp, each breakpoint-defined segment would be about 10 Mbp or aphysical map resolution of about 10 Mbp. The construction of an oat-maize radia-tion hybrid mapping panel diagram and its use to reveal relative relationshipsbetween genetic map distances and physical chromosome segment lengths alongchromosome 1 of maize is shown in Figure 4, which is adapted from data pre-sented in Kynast et al. (2004). Unpublished data in Figure 5 illustrate the use ofa low/medium resolution chromosome 9 radiation hybrid panel to assign a set ofsequences selected from the maize genome database (www.maizegdb.org) to oneof twelve chromosome 9 segments.

435

RINES ET AL.

Rines-4 26-10-2005 9:24 Pagina 435

436

RINES ET AL.

Figure 4. Relative relationships between genetic map distances and physical chromosome segmentlengths along maize chromosome 1. This figure, which is adapted from more detailed data presentedin Kynast et al. (2004), shows on the left the marker content of ten groups of maize chromosome 1radiation hybrids together with that of the parent maize chromosome 1 addition line OMA 1.07.Each group consists of one to five radiation hybrid lines that show similar presence/absence of the 45SSR markers whose order and relative map distance position on the IBM 2 maize genetic linkage mapwww.maizedbg.org) are shown in the center of the figure. On the right are modified chromosomestaken from GISH preparations of root-tip cells of three different radiation hybrid lines in which

Rines-4 26-10-2005 9:25 Pagina 436

RINES ET AL.

437

Figure 5. Physical mapping of maize DNA sequences to chromosome segment using a maize chro-mosome 9 radiation hybrid (RH) panel of nine maize terminal deletion chromosome or oat-maizetranslocation lines that together delineate 12 chromosome segments. Segments 3, 6 and 11 each aredelineated by two RH lines that have a breakpoint between the same SSR markers that were usedto initially characterize the RH lines. The SSR markers are listed across the top of the panel in theirorder on the IBM 2 genetic linkage map. The EST sequences mapped to the RH panel are ones thathad first been mapped to chromosome 9 with a panel of the ten oat-maize addition lines.

fluorochrome-labeled (yellow) total genomic DNA was used as hybridization probe. The chromo-some at the top is from an oat-maize translocation belonging to marker set group 3 in which a ter-minal segment estimated to be about 15% of the short arm of maize chromosome 1 is translocatedonto an oat chromosome. The middle chromosome shown is a maize chromosome 1 missing about20% of its short arm terminus and with marker set group 7. The bottom chromosome has a segmentestimated to be about 20% of the maize chromosome long arm translocated onto an oat chromosomeand belongs to marker set group 9. Comparisons of the sizes of the portions of the genetic mapincluded in the various maize translocation segments, as indicated by the regions marked betweenarrows, provides further evidence in maize for the observation made in numerous large genome plantspecies that the genetic recombination frequency tends to be high toward the ends of chromosomeswhile the central, often more heterochromatic region, is low in recombination. For example, thegroup 3 maize chromosome 1 segment is physically only about 15% of just the short arm butaccounts for about 20% of the total genetic map distance of maize chromosome 1.

Rines-4 26-10-2005 9:25 Pagina 437

The degree of physical map resolution attainable in this oat-maize radiationhybrid system is expected to be below that of the human-rodent somatic cell radia-tion hybrid system upon which it was modeled because of inherent differences in thesystems (Okagaki et al. 2004). The more recent versions of the human-rodent sys-tem allowed physical mapping of more than 30,000 STS sequences to a resolutionof ~100 kb (Olivier et al. 2001). In the oat-maize system the amount of radiationadministered, and hence the frequency of chromosome breakage generated, is limit-ed by the need for the treated seed to germinate and meiotically transmit rearrange-ments. A major advantage of the oat-maize system is that it is whole-plant based withonly meiotically transmissible products recovered. Thus, a large proportion of thelines can be perpetuated indefinitely through generations (Vales et al. 2004).

Another advantage of the oat-maize radiation hybrid system is that it is chro-mosomal-based. This feature is important in that it often enables individual paralogsto be assigned to their respective chromosomal locations in the highly duplicatedmaize genome.

A radiation hybrid physical mapping system for maize should serve to facilitateassemblage of a whole genome map for maize. The value of a radiation hybridapproach is documented with its use in the recently completed assemblage of awhole genome map in the Brown Norway rat (Rat Genome Sequencing Project2004). We are conducting a National Sciences Foundation Plant Genome ResearchProgram funded project to develop a radiation hybrid mapping system for all tenchromosomes of maize (http://agronomy.coafes.umn.edu/cornpep/nsf/index.htm).To date, low resolution maps of 6 to10 segments have been produced for chromo-somes 1, 2, 4, 6 and 9, and medium-high resolution maps of more than 20 seg-ments are being assembled for chromosomes 6 and 9. Certain features of the sys-tem, some possibly reflecting the interactive behavior of specific maize chromo-somes and fragments with their carrier host oat, have appeared as we study additionlines and radiation hybrids for some of the maize chromosomes. These featuresinclude apparent non-random distribution of recovered chromosomal breakpointsin chromosome 3 in contrast to the apparent randomness observed earlier in chro-mosome 9 products (Riera-Lizarazu et al. 2000), widely disparate differencesbetween physical and genetic map distances between markers (Kynast et al. 2004),and occasional problems of mitotic and meiotic transmission stability (Kynast et al.2002, 2004). Still, the widespread use of these unique materials of maize chromo-some additions and radiation hybrids in oat provides exciting opportunities inmaize structural and functional genomics.

Acknowledgements

Based on work supported by the National Science Foundation Grant 011134.

RINES ET AL.

438

Rines-4 26-10-2005 9:25 Pagina 438

References

Bailey JA, Gu Z, Clark RA, Reinert K, Samonte RV, Schwartz S, Adams AD (2002) Recent seg-mental duplications in the human genome. Science 297:1003-1007

Bass HW, Riera-Lizarazu O, Ananiev EV, Bordoli SJ, Rines HW, Phillips RL, Sedat JW, Agard DA,Cande WZ (2000) Evidence for the coincident initiation of homolog pairing and synapsis dur-ing the telomere-clustering (bouquet) stage of meiotic prophase. J Cell Sci 113:1033-1042

Carlson PS, Smith HH, Dearing RD (1972) Parasexual interspecific hybridization. Proc Natl AcadSci USA 69:2292-2294

Comeau A, Nadeau P, Plourde A, Simard R, Maes O, Kelly S, Harper L, Lettre J, Landry B, PierreCA (1992) Media for in vitro ovule culture of proembryos of wheat and wheat-derived inter-specific hybrids or haploids. Plant Sci 81:117-125

Gaut BS (2001) Patterns of chromosomal duplication in maize and their implications for compar-ative maps of the grasses. Genome Res 11:55-66

Jin W, Melo JR, Nagaki K, Talbert PB, Henikoff S, Dawe RK, Jiang J (2004) Maize centromeres:organization and functional adaptation in the genetic background of oat. Plant Cell 16:571-581

Kynast RG, Riera-Lizarazu O, Vales MI, Okagaki RJ, Maquieira SB, Chen G, Ananiev EV, OdlandWE, Russell CD, Stec AO, Livingston SM, Zaia HA, Rines HW, Phillips RL (2001) A completeset of maize individual chromosome additions to the oat genome. Plant Physiol 125:1216-1227

Kynast RG, Okagaki RJ, Rines HW, Phillips RL (2002) Maize individualized chromosomes andderived radiation hybrid lines and their use in functional genomics. Funct Integr Genomics 2:60-69

Kynast RG, Okagaki RJ, Galatowitsch MW, Granath SR, Jacobs MS, Stec AO, Rines HW, PhillipsRL (2004) Dissecting the maize genome by using chromosome addition and radiation hybridlines. Proc Natl Acad Sci USA 101:9921-9926

Laurie DA, Bennett MD (1986) Wheat maize hybridization. Can J Genet Cytol 28:313-316Laurie DA, Bennett MD (1988a) Cytological evidence for fertilization in hexaploid wheat sorghum

crosses. Plant Breed 100:73-82Laurie DA, Bennett MD (1988b) The production of haploid wheat plants from wheat maize cross-

es. Theor Appl Genet 76:393-397Laurie DA, Bennett MD (1989) The timing of chromosome elimination in hexaploid wheat ✕

maize crosses. Genome 32:953-961Laurie DA, O’Donoughue LS, Bennett MD (1990) Wheat maize and other wide sexual hybrids:

their potential for crop improvement and genetic manipulations. In: Gustafson JP (ed) Genemanipulation in plant improvement II, Proc 19th Stadler Genet Symp. Plenum Press, New York,pp 95-126

Li LJ, Arumuganathan K, Rines HW, Phillips RL, Riera-Lizarazu O, Sandhu D, Zhou Y, Gill KS(2001) Flow cytometric sorting of maize chromosome 9 from an oat-maize chromosome addi-tion line. Theor Appl Genet 102:658-663

Meyers BC, Tingey SV, Morgante M (2001) Abundance, distribution, and transcriptional activityof repetitive elements in the maize genome. Genome Res 11:1660-1676

Mochida K, Tsujimoto H (2001) Production of wheat double haploids by pollination with Job’stears (Coix lacrymajobi L.). J Hered 92:81-83

Monfort A, Vicient CM, Raz R, Puigdomenech P, Martinez-Izquierdo JA (1995) Molecular analy-sis of a putative transposable retroelement from the Zea genus with internal clusters of tandemrepeats. J DNA Res 2:255-261

Muehlbauer GJ, Riera-Lizarazu O, Kynast RG, Martin D, Phillips RL, Rines HW (2000) Amaize-chromosome 3 addition line of oat exhibits expression of the maize homeobox gene ligule-

RINES ET AL.

439

Rines-4 26-10-2005 9:25 Pagina 439

less3 and alterations of cell fate. Genome 43:1055-1064Mujeeb-Kazi A, Riera-Lizarazu O (1996) Polyhaploid production in the Triticeae by sexual

hybridization. In: Jain SM, Sopory SK, Veilleux RE (eds) In vitro haploid production in higherplants. Vol 1. Kluwer Academic Publ, Dordrecht, Netherlands, pp 275-296

Okagaki RJ, Kynast RG, Livingston SM, Russell CD, Rines HW, Phillips RL (2001) Mappingmaize sequences to chromosome using oat-maize chromosome addition materials. Plant Physi-ol 125:1228-1235

Okagaki RJ, Kynast RG, Rines HW, Phillips RL (2004) Radiation hybrid mapping. In: GoodmanRM (ed) Encyclopedia of Plant & Crop Science. Marcel Dekker, Inc, New York, pp 1074-1077

Olivier M, Aggarwal A, Allen J, Annalisa Almendras A, Bajorek ES, Beasley EM, Brady SD, BushardJM, Bustos VI, Chu A,Chung TR, De Witte A, Denys ME, Dominguez R, Fang NY, Foster BD,Freudenberg RW, Hadley D, Hamilton LR, Jeffrey TJ, Kelly L, Lazzeroni L, Levy MR, LewisSC, Liu X, Lopez FJ, Louie B, Marquis JP, Martinez RA, Matsuura MK, Misherghi NS, Nor-ton JA, Olshen S,Perkins SM, Perou AJ, Piercy C, Piercy M, Qin F, Reif T, Sheppard K,Shokoohi V, Smick GA, Sun WL, Stewart EA, Tejeda JF, Tran NM, Trejo T, Vo NT, Yan SCM,Zierten DL, Zhao S, Sachidanandam R, Trask BJ, Myers RM, Cox DR (2001) A high-resolu-tion radiation hybrid map of the human genome draft sequence. Science 291:1298-1302

Rat Genome Sequencing Project Consortium (2004) Genome sequence of the Brown Norway ratyields insights into mammalian evolution. Nature 428:493-521

Riera-Lizarazu O, Mujeeb-Kazi A (1990) Maize (Zea mays L.) mediated wheat (Triticum aestivumL.) polyhaploid production using various crossing methods. Cereal Res Commun 18:339-346

Riera-Lizarazu O, Mujeeb-Kazi A (1993) Polyhaploid production in the Triticeae: wheat Tripsacumcrosses. Crop Sci 33:973-976

Riera-Lizarazu O, Rines HW, Phillips RL (1996) Cytological and molecular characterization of oat-maize partial hybrids. Theor Appl Genet 93:123-135

Riera-Lizarazu O, Vales MI, Ananiev EV, Rines HW, Phillips RL (2000) Production and charac-terization of maize-chromosome 9 radiation hybrids derived from an oat-maize addition line.Genetics 156:327-339

Rines HW (1983) Oat anther culture: Genotype effects on callus initiation and the production ofa haploid plant. Crop Sci 23:268-272

Rines HW, Dahleen LS (1990) Haploid oat plants produced by application of maize pollen toemasculated oat florets. Crop Sci 30:1073-1078

Rines HW, Riera-Lizarazu O, Phillips RL (1995) Disomic maize chromosome-addition oat plantsderived from oat maize crosses. In: Oono K, Takaiwa F (eds) Modification of gene expressionand non-Mendelian inheritance. National Institute of Agrobiological Resources, Tsukuba,Japan, pp 205-221

Ruddle FH (1973) Linkage analysis in man by somatic cell genetics. Nature 242:165-169.Sharma HC, Gill BS (1983) Current status of wide hybridization in wheat. Euphytica 32:17-31Suenaga K, Nakajima K (1989) Efficient production of haploid wheat (Triticum aestivum) through

crosses between wheat and maize (Zea mays). Plant Cell Rep 8:263-266Szarka B, Gonter I, Molnar-Lang M, Morocz S, Dudits D (2002) Mixing of maize and wheat

genomic DNA by somatic hybridization in regenerated sterile maize plants. Theor Appl Genet105:1-7

Taketa S, Choda M, Ohashi R, Ichii M, Takeda K (2002) Molecular and physical mapping of a bar-ley gene on chromosome arm 1H that causes sterility in hybrids with wheat. Genome 45:617-625

Ushiyama T, Shimizu T, Kuwabara T (1991) High frequency of haploid production of wheatthrough intergeneric cross with Teosinte. Jpn J Breed 41:353-357

RINES ET AL.

440

Rines-4 26-10-2005 9:25 Pagina 440

Vales MI, Riera-Lizarazu O, Rines HW, Phillips RL (2004) Transmission of maize chromosome 9rearrangements in oat-maize radiation hybrids. Genome 47:1202-1210

Zenkteler M, Nitzsche W (1984) Wide hybridization in cereals. Theor Appl Genet 68:311-315

RINES ET AL.

441

Rines-4 26-10-2005 9:25 Pagina 441