A Maize Map Standard With Sequenced Core Markers, Grass Genome Reference Points and 932 Expressed Sequence Tagged Sites (ESTs) in a 1736-Locus Map

Corresponding author: E. H. Coe, Jr., USDA-ARS, MWA, PGRU, 210 Curtis Hall, Columbia, MO 65211., ed{at}teosinte.agron.missouri.edu (E-mail)

Communicating editor: W. F. SHERIDAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have constructed a 1736-locus maize genome map containing1156 loci probed by cDNAs, 545 probed by random genomic clones, 16 by simple sequence repeats (SSRs), 14 by isozymes, and 5 by anonymous clones. Sequence information is available for 56% of the loci with 66% of the sequenced loci assigned functions. A total of 596 new ESTs were mapped from a B73 library of 5-wk-old shoots. The map contains 237 loci probed by barley, oat, wheat, rice, or tripsacum clones, which serve as grass genome reference points in comparisons between maize and other grass maps. Ninety core markers selected for low copy number, high polymorphism, and even spacing along the chromosome delineate the 100 bins on the map. The average bin size is 17 cM. Use of bin assignments enables comparison among different maize mapping populations and experiments including those involving cytogenetic stocks, mutants, or quantitative trait loci. Integration of nonmaize markers in the map extends the resources available for gene discovery beyond the boundaries of maize mapping information into the expanse of map, sequence, and phenotype information from other grass species. This map provides a foundation for numerous basic and applied investigations including studies of gene organization, gene and genome evolution, targeted cloning, and dissection of complex traits.

MAIZE research has had a long tradition in the area of gene mapping. The first published genetic map compiled by EMERSON et al. 1935 contained 62 loci based on morphological variants. Refinement of this map progressed on the basis of accumulated recombination data for the next 60 years. A cytological map based on B-A translocations (ROMAN and ULLSTRUP 1951 ) and phenotypic markers is also available (BECKETT 1991 ). The use of B-A and reciprocal A-A translocations permitted the physical map to be oriented and aligned with the genetic map. The first molecular-marker maize map was published by HELENTJARIS et al. 1986 . It contained 116 loci and used cDNA and random genomic clones as probes. Maps based on publicly available maize restriction fragment length polymorphism (RFLP) probes were presented by COE et al. 1987 and BURR et al. 1988 using random genomic clones on an F₂ population and on a set of recombinant inbred lines, respectively. GARDINER et al. 1993 published an updated version of the earlier F₂ map on the basis of an immortalized version of the same F₂ population. It contained 214 loci and the first group of "core" markers. WEBER and HELENTJARIS 1989 used RFLP analysis of progeny from crosses of B-A translocation lines with inbreds to link the cytological map information with the molecular map. BEAVIS et al. 1992 developed a mapping population based on random mating that provided improved resolution compared to F₂ or recombinant inbred populations of similar size. More recently, a composite map based on four mapping populations, containing 275 loci representing both expressed sequence tagged sites (ESTs) and anonymous sequences was published (CAUSSE et al. 1996 ). A number of other groups have produced RFLP maps in maize, most with the intent of mapping quantitative traits relative to the molecular markers (BEAVIS and GRANT 1991 ; EDWARDS et al. 1992 ; PE et al. 1993 ; AJMONE-MARSAN et al. 1994 ; DAMERVAL et al. 1994 ; FROVA and SARI-GORLA 1994 ; QUARRIE et al. 1994 ; SARI-GORLA et al. 1994 ; VELDBOOM and LEE 1994 ; VELDBOOM et al. 1994 ; CAUSSE et al. 1995 ; LEBRETON et al. 1995 ; AGRAMA and MOUSSA 1996 ; AUSTIN and LEE 1996A , AUSTIN and LEE 1996B ; BOHN et al. 1996 ; BYRNE et al. 1996 ; LUBBERSTEDT et al. 1997 ). PCR-based DNA markers composed of tandemly repeated short di- or trinucleotide repeats known as simple sequence repeat (SSR) markers have also been utilized to map genes in maize (SENIOR and HEUN 1993 ; TARAMINO and TINGEY 1996 ). These markers are more amenable to high-throughput mapping but are not yet available in sufficient number to provide even coverage of the maize genome.

We present here a linkage map containing a large number of ESTs and sequence-tagged sites (STSs), a set of 90 core markers, and 237 loci probed by clones from other grass species. These three features allow the information from this map to be combined with other maize and grass species map data to facilitate a variety of gene discovery experiments.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Laboratory procedures:
DNA was prepared using the mixed alkyltrimethyl-ammonium bromide (CTAB) extraction method (SAGHAI-MAROOF et al. 1984 ) from 54 immortalized F₂ individuals from a cross of Tx303 x CO159, the inbred parents and their F₁ hybrid (GARDINER et al. 1993 ). Restriction digestions were performed using EcoRI, HindIII, EcoRV, BamHI, DraI, XbaI, BglII, or SstI. DNA was electrophoresed in 0.8% agarose gels at 50 V for a distance of ~10 cm. Gels were denatured for 30 min in 0.4 N NaOH, 0.6 M NaCl followed by a 30-min neutralization in 0.5 M Tris, pH 7.5, 1.5 M NaCl. DNA was transferred to Magnacharge membrane using 25 mM NaPO₄, pH 6.5. Membranes were baked and cross-linked according to manufacturer recommendations (MSI, Westborough, MA).

Probes used in this study included cDNA and genomic clones from maize and other related grasses. They included agr maize clones (Mycogen Plant Sciences), asg maize clones (Asgrow Seed), bnl maize clones (Brookhaven National Laboratory), csu maize clones (Chris Baysdorfer, California State University-Hayward), npi maize clones (Native Plants & Pioneer Hi-Bred International), php maize clones (Pioneer Hi-Bred International), uaz maize clones (Tim Helentjaris, University of Arizona), umc maize clones (University of Missouri-Columbia), rgp rice clones (Rice Genome Research Project, Tsukuba, Japan), bcd barley clones (Susan McCouch, Cornell University), cdo oat clones (Susan McCouch, Cornell University), rz rice clones (Susan McCouch, Cornell University), umn oat clones (Ronald Phillips, University of Minnesota), tda tripsacum clones (Ann Blakey, Ball State University), and numerous genes from targeted cloning experiments supplied by individual investigators.

Membranes were prehybridized a minimum of 6 hr in a solution of 0.05 M Tris, pH 8.0, 0.01 M EDTA, pH 8.0, 5x SSC, 0.2% SDS, 1x Denhardt's solution, 0.1 mg/ml denatured salmon sperm DNA. All hybridizations were carried out using [-³²P]dCTP oligo-labeled probes at 65° overnight in a hybridization solution containing the above ingredients plus 10% dextran sulfate. Washing protocol was as follows: three 5-min room temperature washes in 2x SSC, 0.5% SDS, one 20-min room temperature wash in 0.1x SSC, 0.1% SDS, and two 30-min 65° washes in 0.1x SSC, 0.1% SDS. Membranes were patted dry with toweling, placed in plastic sheet protectors, and exposed to Kodak X-OMAT film in the presence of a CRONEX-type intensifying screen for 1 to 7 days at -80° depending on the counts per minute as determined with a Ludlum-III monitor (Sweetwater, TX).

The enzyme with the best fragment separation between the two parental lines, CO159 and Tx303, was chosen for mapping. In some cases, for multiple copy probes, more than one enzyme was used to map additional loci.

The polymerase chain reaction (PCR) conditions and cycling profiles for SSR analysis were based on the protocol established by M. LYNN SENIOR (personal communication) but included slight modifications, mainly to accommodate the specific polymerase used for the experiment. Final concentrations of reaction components were as follows: Perkin-Elmer Buffer, 1x; MgCl₂, 2.5 mM; dATP, dCTP, dGTP, dTTP, 0.1 mM each, SSR primers-forward and reverse, 50 ng each (Research Genetics, Huntsville, AL); AmpliTaq Gold polymerase, 0.3 units (Perkin-Elmer, Norwalk, CT); genomic DNA, 50 ng; sterile water to a total volume of 15 µl.

All thermocycling was performed in a 96-well thin-walled microtiter-style plate [Costar (Cambridge, MA) 6509] with an oil overlay in an Amplitron II thermocycler (Barnstead/Thermolyne, Dubuque, IA). The cycling profile included a preliminary 8- to 10-min dwell at 95° to activate the polymerase. This was followed by two cycles of 1 min at 95°, 1 min at 65°, and 90 sec at 72°. Subsequently, single cycles of a 1° decrement for the annealing temperature were done until an annealing temperature of 55° was achieved. The final phase of amplification included 29 additional cycles at the 55° annealing temperature. Following amplification, PCR products were resolved in a 3.5% Super Fine Resolution agarose (Amresco Inc., Solon, OH), 1x TBE gel containing ~2 µg/ml ethidium bromide.

Data collection and map construction:
All autoradiograms were scored independently by two readers. SSR gel images were captured with a CCD camera system (Stratagene, La Jolla, CA) and genotypes were recorded from either computer monitor images or thermal prints. Markers with missing data for three or more individuals were typically discarded.

Linkage groups were constructed using MAPMAKER for UNIX, version 3 (Whitehead Institute, Cambridge, MA) on a Sun SPARC Server 1000 (Sun Systems, Palo Alto, CA). The 10 maize linkage groups were defined with the "make chromosomes" function and the 90 core markers were anchored to linkage groups. Initial framework orders were assigned for the core markers for each linkage group on the basis of previous map constructions at LOD 5 for chromosomes 1 and 3–10 and LOD 4 for chromosome 2 (COE et al. 1995 ). The remaining markers were attached to linkage groups with the "assign" command. Additional markers were three-point ordered into the framework, first at LOD 3 and then at LOD 2, 10–15 markers at a time with the "build" command. Remaining markers assigned to each linkage group were positioned relative to the framework loci with the "place" command. Loci with unique positions were inserted into the framework while those lacking sufficient recombination events to provide three-point order were positioned on the basis of two-point analysis. The "together" command was used to further resolve the position of the "placed" loci in the framework. The remaining markers were then positioned against the new framework with the "place" command. Marker loci with more than three double crossovers based on the "genotype" function were deleted. Three-point local ordering was assessed using the ripple command in 5-marker intervals with a threshold LOD of 3.0. Significantly better alternate orders were identified for two intervals tested. Ten additional regions were identified where alternate orders were available at LOD 3.0. For these 10 regions, numbers of single and double crossovers for each alternate order were assessed. In 6 of the 10 cases, double crossovers were minimized by one of the alternate orders and map orders were set to reflect the order that minimized double crossovers. In the 4 remaining cases, two three-point orders were equally likely. These regions are asg29b-csu2a-csu850-umc8g vs. asg29b-umc8g-csu850-csu2a (bin 2.05), csu587b-csu26a-umc108-asg84b vs. csu587b-umc108-csu26a-asg84b (bin 5.07), csu835-csu481-csu360-csu116a vs. csu835-csu360-csu481-csu116a (bin 6.02), and umc238a-idh2-csu291-mdh2 vs. umc238a-csu291-idh2-mdh2 (bin 6.07). Chromosome maps were drawn to postscript files, exported, translated to Macintosh format, and edited with Adobe Illustrator 6.0.1 (Adobe Systems, Mountainview, CA).

The core marker set:
Markers that had simple fragment patterns and were distributed along the chromosome approximately every 20 cM were selected as potential core markers. Markers not among the previous core set identified by GARDINER et al. 1993 were screened against inbreds A619, A632, B73, Mo17, CO159, and Tx303 using EcoRI, HindIII, EcoRV, BamHI, DraI, XbaI, BglII, and SstI restriction endonucleases to determine whether they were polymorphic enough to be designated as core markers. A marker was deemed to be acceptable if it was polymorphic with a minimum of three of the eight enzymes with the majority of inbred lines. Subsequently, all the previous core markers were screened in the same manner. Final choices were made on the basis of even-spacing, simple-fragment patterns, high degree of polymorphism, and public availability. Markers meeting these criteria that had insert sizes <1000 bp were given preference to facilitate more complete single-pass sequencing of the clones.

The csu clones:
Sequencing of csu clones through number 173 was reported previously (KEITH et al. 1993 ). The 5'-ends of clones csu174 through csu1196 were dideoxy-sequenced according to the protocol of KEITH et al. 1993 . These clones were selected from a B73 library of 5-wk-old shoots. Sixty-six clones that were duplicates of other csu clones in this group on the basis of sequence comparisons among group members were removed from the clone set prior to screening and mapping. DNA sequence data were submitted to GenBank for all nonduplicate clones.

Core marker sequencing:
Core marker insert DNA maintained in plasmids was prepared for sequencing by the alkaline lysis method (BIRNBOIM 1983 ). DNA quality was determined using a 0.8% agarose gel containing cut and uncut DNA. The quantity of DNA was determined using a spectrophotometer for samples with acceptable quality. Sequencing was performed one of two ways. In the first case, dideoxy-termination reactions labeled with [-³⁵S]dCTP were performed according to manufacturer recommendations using the T7 Sequenase Kit (U.S. Biochem, Cleveland). Depending upon the vector the cloned RFLP was ligated into, M13 forward and reverse or T7 and SP6 primers were used to sequence the DNA. For each direction, 2 µg of dsDNA was annealed to the appropriate primer. Reactions were run on a 6% polyacrylamide gel for 2 hr for short runs to read sequence close to the primer or 6 hr for long runs. Gels were fixed in 10% acetic acid/5% methanol and thoroughly dried under vacuum. Dried gels were exposed to Kodak X-OMAT AR film for 1 to 2 days. Sequence data were double checked. In the second case, sequencing was done by PCR incorporation of fluorescently labeled bases followed by data generation on the ABI 373 sequencing machine (PE Applied Biosystems, Foster City, CA).

Homology searching:
Sequence similarity data were provided by individual investigators with each cDNA or genomic clone from targeted cloning experiments submitted for mapping at the Maize RFLP Laboratory. Following map construction and refinement, all noncore sequences that were not received from targeted cloning experiments were analyzed for homology using the NCBI blast server or the dbEST neighbors algorithm (www.ncbi.nlm.nih.gov/BLAST; ALTSCHUL et al. 1990 ; http://www.ncbi.nlm.nih.gov/irx/dbST/dbest_query.html; http://www.ncbi.nlm.nih.gov/Entrez/entrezhelp.html#Special; BOGUSKI et al. 1993 ). Following BLASTX searching, sequences with P values of <10^-8 against the EST database, 10^-10 against the nonredundant nucleotide or nonredundant peptide databases, or 10^-10 using the neighbors algorithm were assigned putative functions. Mnemonics were derived for the loci associated with those clones that were consistent with the maize nomenclature guidelines (www.agron.missouri.edu/maize_nomenclature.html) and using plantwide nomenclature (http://jiio6.jic.bbsrc.ac.uk) whenever possible. In assignments both strands of genomic sequences were considered, while only the positive strand of directionally cloned cDNAs was considered. At the time the comparisons were conducted, the nonredundant nucleotide database did not include EST or STS sequences but did include GenBank, DDBJ, EMBL, and Protein Data Bank (PDB) sequences; the nonredundant peptide database included translations of the GenBank coding sequences and sequences in SwissProt, PDB, and Protein Information Resource (PIR).

Core marker sequences were analyzed using the e-mail version of BLAST1.4.11 (ALTSCHUL et al. 1990 ; blast@ncbi.nlm.nih.gov). Homology searches against the NCBI nr and dbEST databases were made using, respectively, the BLASTX and TBLASTX algorithms. Identity was declared at P(N) <10^-6. In some cases, although a gene name is given, it was clear that the reported match is only to a motif, not to the entire gene.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

EST Map:
A map containing 1736 loci was produced (Figure 1). This represents an increase of 1427 loci over the previously published map (CHAO et al. 1994 ). The total maize map length was 1727.4 cM, with one crossover equal to 0.9 cM. Chromosome 1 had the longest map distance at 245.2 cM and chromosome 10, the shortest at 138.6 cM (Table 1). In general, chromosome length as measured genetically decreased with decreasing physical length from chromosome 1 through 10. Chromosome 7 was a noticeable exception to this trend. The largest remaining gap (22.8 cM) in the maize map occurs in the telomere region of chromosome 7. Eleven other gaps of >10 cM occur in telomeric regions. Twelve additional gaps of >10 cM occur throughout the internal regions of the chromosomes.

View larger version (325K): [in this window] [in a new window] [Download PPT slide]   Figure 1. UMC EST map of Zea mays L. The map was constructed using 54 immortalized F2 individuals derived from Tx303 x CO159. Markers are listed to the right of the map line. Large numbers (chromosome number, bin number, i.e., 1.01) to the left of the chromosome indicate the chromosome bins. Core marker loci are shown in boxes immediately to the right of each hash line on the chromosome and represent the first marker in the bin below. Interval support for these markers is LOD 5 for all but chromosome 2, which is LOD 4. Numbers to the left of the hash line are coordinates. Small numbers immediately to the left of the chromosome indicate centimorgan distances between the markers using Haldane's correction. Loci in bold are set to the framework and order assured at LOD 2.0. Italics indicates loci that are placed by two-point association to the boldfaced loci on the same line; order for these loci is uncertain but placement can be considered to be at or below the framework mark at which they are shown, on the basis of tests using alternative orders of frameworked markers. Estimated centromere locations are represented by a wider line, on the basis of composite judgment of mapping data for genes and molecular markers, as well as B-A translocations and other cytogenetic information contained in MaizeDB (http://www.agron.missouri.edu). Acronyms for loci probed by nonmaize clones are indicated as follows: bcd (barley), cdo (oat), rgp (rice), rz (rice), tda (tripsacum), and umn (oat). URLs for tabular versions of each linkage group are provided below the total centimorgan values.

Of the 1736 loci, 1156 (67%) were probed by cDNAs, 545 (31%) were probed by genomic clones, 14 (1%) corresponded to isozymes, 16 (1%) were mapped using SSRs, and 5 represented anonymous clones. Of the probes screened 19% were single copy, 58% medium copy (2–5 copies), and 23% high copy number (>5 copies). A total of 590 new ESTs from the csu clone set were mapped. The total number of loci per chromosome decreased from longest to shortest chromosome with the exception of chromosomes 2, 6, and 7, which had fewer markers than expected (Table 1).

Sequence homology:
Table 2 lists the loci for which sequence homology has been determined, the database entry they matched, and maize bin location. GenBank numbers are provided for csu clones sequenced and mapped as part of this study. The data indicate that 637 (36.7%) of all loci corresponded to genes of known function. Of the loci on the map 56% (962) have sequence information available. The designated genes and loci with sequence information are fairly evenly distributed among the chromosomes (Table 1). Of the loci with sequence information, 66% have been assigned putative functions with only 34% having no known function at this time. Nucleotide matches were identified to regulatory factors as well as structural genes and included such diverse processes as membrane transport, signal transduction, cell cycle regulation, carbon metabolism, floral development, stress response, DNA synthesis, and fatty acid metabolism.

Of the loci corresponding to previously unpublished csu sequences 41% (242) had homology to known genes. Among the new csu clones, 41 were single copy and were given gene designations. Several of the new csu sequences corresponded to isozymes previously mapped using protein gels: p-csu262, corresponding to pgd1 and pgd2, which encodes 6-phosphogluconate dehydrogenase; p-csu892, encoding ADP-glucose pyrophosphorylase (agp2); p-csu249, encoding malate dehydrogenase (mdh5); p-csu182, encoding superoxide dismutase (sod4); and p-csu301, encoding triose phosphate isomerase (tpi4). No recombinants were detected between the isozyme and cDNA mapscores for pgd1 in this population. pgd2, mdh5, and tpi4 were previously mapped as isozymes on the Brookhaven National Laboratory (BNL) map. Common flanking markers between the two maps were used to confirm the common positions of the isozymes and cDNAs. Relative map positions of isozymes and cDNAs were determined for agp2 and sod4 on the basis of alignment of this map with the classical genetic map using common RFLP and isozyme markers as a bridge.

Core markers:
Ninety core markers were identified that best met the selection criteria of low copy number, high rate of polymorphism, even spacing, and public availability. Core marker interval support was LOD 5 for all but maize chromosome 2, which had interval support of LOD 4. The lower LOD support for chromosome 2 is a reflection of the larger size of bin 2.02. Forty of the previous core markers were retained but several substitutions were made to the previous core marker set (Table 3). The core markers delineate 100 bins with the average bin size equaling 17 cM. Polymorphism information for the inbreds and enzymes screened is available in the Maize Genome Database (MaizeDB, http://www.agron.missouri.edu). Core marker sets are available via the probe request hotlink in MaizeDB or by contacting the University of Missouri–Columbia Maize RFLP Laboratory c/o Theresa Musket.

Sequence data were obtained for 84 of the core markers. Table 3 contains the sequence similarity information and GenBank numbers for the core markers. Homology to genes of known function was identified for 14 of the core markers by BLASTX searching of the nonredundant database. Matches to functionally uncharacterized ESTs or unknown proteins were identified for 7 additional core markers. Functionality of four of the clones had been identified by targeted cloning experiments. Seventeen percent of the genomic cores and 11% of the cDNA cores derived by nontargeted cloning had matches to genes of known functions.

Grass genome reference points:
The map contains 25 loci probed by barley clones, 56 by oat clones, 136 by rice clones, 19 by tripsacum clones, and 1 by a wheat clone, for a total of 237 loci probed by nonmaize clones. The majority of the clones are anchor markers from the Cornell grass maps or from the Japanese Rice Genome Project (RGP) map. There are 221 loci probed by maize or rice clones shared between this map and the RGP rice map (HARUSHIMA et al. 1998 ).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The map presented here provides a more than fivefold increase in number of loci compared to the map previously published in this population (CHAO et al. 1994 ). It is slightly smaller than the total map lengths of 1883.6 cM for the 1995 Brookhaven maize map (MATZ et al. 1995 ) and 1765 cM for the INRA maize map (CAUSSE et al. 1996 ). The smaller population used to construct this map, in comparison with that used for the BNL and INRA maps, may be partially responsible for the difference in map length, as fewer recombinants would be expected in the smaller sample. This difference may also be the result of fewer telomere loci, which often show high rates of recombination, being located on the present map (four compared to eight on the Brookhaven map), and indicates that mapping additional telomeres would be desirable. The map presented here is substantially smaller than the 1996 BNL maize map, which has a total length of 2048.6 cM (B. BURR, personal communication; ftp://ftp.bio.bnl.gov/pub/maize/chrom.map). The remaining gaps are likely to represent regions of small physical vs. large genetic distance rather than marker-poor regions because increased rates of recombination are observed in telomeric regions where many of the gaps of >10 cM occur. Syntenic analysis of maize and rice indicates that the remaining internal gaps are also likely to be regions of small physical vs. large genetic distance, suggesting that all chromosomes are adequately marked (G. DAVIS, unpublished results).

Given the rate at which sequence information is being generated and the resolution ability of this and other current mapping populations, a threshold has been reached where future local ordering will be unfeasible and/or impractical. Precise local order and distance information is a necessary component of many gene discovery experiments. Therefore, alternative types of mapping populations are a requirement for advances in plant genetics in the near future. The current automated sequencing technologies have accelerated the pace at which unique clones can be identified to the point where the increases of population size in F₂, recombinant inbred, or backcross populations required to order tightly linked markers will no longer be feasible or economical. Development of radiation hybrid mapping panels (COX et al. 1990 ), physical approaches such as YAC or BAC contigs (SCHMIDT et al. 1995 ; ZACHGO et al. 1996 ; ZHANG and WING 1997 ), or DNA microchip arrays for genome scanning (NELSON et al. 1993 ; SHALON et al. 1996 ) currently provide the best prospects to meet the needs of maize researchers.

Four features combined to heighten the utility of this map as a foundation for basic and applied investigations in such areas as gene organization, gene and genome evolution, cloning of single genes, and dissection of complex traits. They are the use of a variety of probe types, availability of sequence information for a majority of the loci, designation of a group of core markers, and mapping of grass genome reference points.

Varied probe types:
In this study we have utilized both cDNA and genomic clones as probes. Initial maize maps were based primarily on random genomic DNA probes, which have the advantage of being relatively straightforward and rapid to make. Most genomic probes have been derived by cutting hypomethylated regions with PstI. The copy number is usually lower for genomic than cDNA probes, possibly because of exon or 5'/3'-UTR divergence of the genomic fragments. Given the chromosomal duplication present in the maize genome, genomic probes are often advantageous as reference markers in comparisons between different maize mapping populations. Because many of the genomic clones are single copy, it is easy to infer position information from one genetic background to another because there is no confusion regarding which fragment corresponds to which locus. Use of ESTs as probes for mapping, on the other hand, facilitates the association of functionality with phenotype—for example, coincidental location of an EST having a known product with a phenotypic mutant with the appropriate developmental or biochemical defect (see examples later in this discussion). In addition to traditional hybridization-based markers, several SSR loci have been mapped. SSRs offer a more rapid, radiation-free alternative to hybridization-based marker technologies. Although these markers are more easily adapted to high-throughput mapping, further development in maize is needed to generate enough markers to provide thorough coverage of the entire genome in diverse genetic backgrounds.

Data from 1383 markers that have been mapped in rice indicate that 33% of the rice markers were single copy and another 31% were "near single copy" (KURATA et al. 1994 ). These percentages are much greater than the 19% for single-copy markers observed in this study. The higher hybridization temperatures used in the maize study would favor lower copy number of clones in maize relative to rice if hybridization conditions were responsible for the observed difference in magnitude of single-copy clones. The difference between maize and rice in percentage of low-copy-number markers likely reflects the chromosomal duplication present in the maize genome (WENDEL et al. 1986 ; HELENTJARIS et al. 1988 ).

Sequence availability:
Only 34% of the loci with sequence information remain functionally uncharacterized by the criteria used in this experiment. This is encouraging, both because a high percentage of loci with sequence information could be tentatively assigned functions and because the number of loci without putative function should decrease with time as additional information regarding sequence and function is made available through the public sequence databases. As of July 20, 1998, Arabidopsis, rice, and maize represent only 3.9% of the 1.7 million sequences in dbEST. To continue to fully utilize the molecular map as a discovery and development tool, additional plant cDNAs must be sequenced and mapped. Sequence information is fundamental in unraveling the relationship of biochemical processes to developmental and agronomic characteristics. The sequence from a given maize cDNA can be searched against a continuously expanding library of sequence information within maize, among other plant species, and more broadly among the animal and human sequences that are publicly available. Matches can be used in an attempt to assign functionality to a given phenotype.

Although the number of maize genes sequenced is much lower than that of Arabidopsis genes, the number of loci mapped is comparable and the percentage with putative function is greater. The Arabidopsis EST project has sequenced ~10,000 distinct genes, representing about half of the estimated total gene number (DELSENY et al. 1997 ). Of those, 40% have putative function and 1500 ESTs have been placed on the physical map. The comparison with rice is somewhat different. More than 10,500 unique sequences have been identified representing approximately one-third of all the rice genes; however, only 25% of the clones have significant protein similarity (YAMAMOTO and SASAKI 1997 ). The lower similarity compared to maize and Arabidopsis may reflect the differences in number and types of tissues used to construct the cDNA libraries in each organism. Approximately 2300 DNA markers, including 2000 RFLP markers, have been mapped on the RGP rice map (NAGAMURA et al. 1997 ). The number of loci mapped in any of the plant species pales by comparison to the more than 10,400 loci mapped in a single STS map of the human genome (STEWART et al. 1997 ). Recent estimates indicate that approximately one-half of the total genes in the human genome have been sequenced and that only ~21% of these have significant similarity to a known protein (SCHULER et al. 1996 ). It is speculated that the lower percentage of putative functions identified for the human genome sequences is the result of bias introduced by ESTs that contain 3'-untranslated regions that are not protein encoding and thus are not capable of matching known proteins.

Constructing a molecular map containing expressed sequence-tagged sites is the first step toward generating an expression map for an organism. This information can be used to examine tissue, organ, and/or developmental specificity of gene expression for individual members of a multigene family. Alternatively, it can be used to examine the pattern of gene expression along a particular region of a chromosome to answer such questions as, "are the genes for a particular developmental event clustered on the chromosome?" Additional information to be gained includes common motifs or higher order structures obtained through comparison of promoter regions from different genes expressed in the same tissue or organ or at the same developmental time.

Core markers:
The entire set of core markers and their sequences are publicly available. This enables the chromosomal regions identified as significant contributors to qualitative and quantitative traits to be assigned to bins on the same framework, referred to as the bin map. Alignment of RFLP, genetic, and cytological maps can also be made on the basis of the framework established by the core markers. The five csu clones that encode isozymes previously placed by phenotype on the genetic map provide new reference points for integrating the molecular and classical genetic maps. Additional ESTs from the Brookhaven and INRA maize maps (BURR et al. 1996 ; CAUSSE et al. 1996 ) can be assigned relative map positions, thereby enhancing the number of loci with information about function. The 376 common loci that exist between this map and the BNL map will provide for alignment among maize molecular maps. Currently, a resource of 5179 loci that have been catalogued to bin is available in MaizeDB.

The combination of sequence and function information for an increasing proportion of the loci on the map, coupled with cataloguing of loci into bins delineated by the core markers, opens the door for identification of potential associations of functions with phenotypes by coincidence, serendipity, and concurrence. One such example involves clone p-csu186, a single-copy cDNA that maps to bin 2.02. Sequence similarity information indicates this clone has homology to ent-kaurene synthase B from Cucurbita maxima. Examination of phenotypic mutants present in bin 2.02 identified a dwarf mutant, d5, that has been biochemically characterized as a defect in cyclization to ent-kaurene synthase B (PHINNEY 1984 ). The coincidental location of a putative ent-kaurene synthase B cDNA with a phenotypic mutant altered in the same biochemical reaction illustrates the potential of utilizing the maize bins to group molecular and phenotypic information to facilitate molecular dissection of traits.

Grass genome reference points:
Several investigators have identified a colinear relationship among loci in members of the grass family (HULBERT et al. 1990 ; WHITKUS et al. 1992 ; BENNETZEN and FREELING 1993 ; MOORE et al. 1993 ). Alignment of sequence data and map locations across organisms will become an increasingly important aspect of future discovery and development strategies. To facilitate this alignment, 237 loci identified with nonmaize probes have been included in the map to provide grass genome reference points. Alignment of common markers on maps of other members of the grass family with those on the maize map allows tentative assignment of additional functionality to the maize map by relative placement of loci not mapped in maize onto the maize bin map. This type of electronic mapping will speed the progress of gene discovery while reducing costs. Such electronic mapping is not without caveats. Although colinearity among grasses exists for large genomic regions, chromosomal rearrangements and genome divergence necessitate a moderately high number of reference points between species before a meaningful integration of information can be achieved between organisms. Once integrated, the markers represent a tentative assignment of additional functions to a particular bin because of potential differences in gene copy number and tissue specificity that have occurred during the course of speciation and evolution. Currently, 221 loci are shared with the RGP rice map, 237 loci with the Cornell University map, and a similar number with the sorghum map. The loci catalogued to bin will be expanded in the near future by the addition of EST and phenotypic loci from analogous regions in rice (G. DAVIS, unpublished results).

Just as in the case cited earlier of coincidental function and phenotype within maize, sequence homology information and mapping information from different species can be combined to provide a powerful discovery tool for comparisons of function vs. phenotype. An example of this involves a candidate cDNA, p-csu838, and a phenotype represented by a QTL for days to pollen shed. This cDNA has homology to a LUMINIDEPENDENS protein from Arabidopsis that corresponds to an Arabidopsis mutant that displays a late-flowering phenotype. The maize cDNA maps to bin 3.05. QTL data from a tropical maize population collected at two locations indicate that a QTL for days to pollen shed also occurs in bin 3.05 (CIMMYT 1994 ). This example shows a potential link between phenotypes from a dicot and a monocot on the basis of sequence homology and relative map position.

Colinearity combined with homology information leads to the next phase of discovery: comparing function, expression, and phenotype across species. This increases the power to determine what makes each species unique and how evolutionary forces work to shape a new species.

The information presented here coupled with map and sequence information from other maize studies and other grass species provides a strong foundation upon which to build an integrated understanding of sequence, biochemical and metabolic functions, and phenotypic effect. Future large-scale sequencing and mapping efforts will expand on these discovery tools.

	FOOTNOTES

¹ Present address: USDA-ARS, Midsouth Area, Corn Host Plant Resistance Research Unit, 117 Dorman Hall, Mississippi State, MS 39762.
² Present address: USDA-ARS, Midwest Area, Corn Insect and Crop Genetics Research Unit and Iowa State University, Ames, IA 50011.
³ Present address: Myriad Genetics Labs, Salt Lake City, UT 84108.
⁴ Present address: Centre Plant Conservation Genetics, Southern Cross University, Lismore NSW 2480, Australia.

	ACKNOWLEDGMENTS

This research was supported by National Research Initiative Grant 94-37300-0329 and U.S. Department of Agriculture-Agricultural Research Service (ARS) CRIS 3622-21000-011-00D. The authors wish to thank the firms and numerous individuals of the maize genetics community who provided clones used for mapping in this study. Maize marker sets were provided by Mycogen Plant Sciences (agr), Asgrow Seed (asg), Brookhaven National Laboratory (bnl), Chris Baysdorfer, California State University-Hayward (csu), Native Plants & Pioneer Hi-Bred International (npi), Pioneer Hi-Bred International (php), Tim Helentjaris, University of Arizona (uaz), and University of Missouri-Columbia (umc). Nonmaize marker sets were provided by the Rice Genome Research Project, Tsukuba, Japan (rgp), Susan McCouch, Cornell University (bcd, cdo, rz), Ronald Phillips, University of Minnesota (umn), and Ann Blakey, Ball State University (tda). Thanks to Shirley Kowalewski and Beth Bennett for assistance in preparation of the map figure and to Sukumar Saha and Karen Cone for their critical evaluation of the manuscript. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.

Manuscript received September 18, 1998; Accepted for publication March 29, 1999.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

AGRAMA, H. A. S. and M. E. MOUSSA, 1996  Mapping QTLs in breeding for drought tolerance in maize (Zea mays L.). Euphytica 91:89-97.

AJMONE-MARSAN, P., G. MONFREDINI, W. F. LUDWIG, A. E. MELCHINGER, and P. FRANCESCHINI et al., 1994  Identification of genomic regions affecting plant height and their relationship with grain yield in an elite maize cross. Maydica 39:133-139.

ALTSCHUL, S. F., W. GISH, W. MILLER, E. W. MYERS, and D. J. LIPMAN, 1990  Basic local alignment search tool. J. Mol. Biol. 215:403-410[Medline].

AUSTIN, D. F. and M. LEE, 1996a  Genetic resolution and verification of quantitative trait loci for flowering and plant height with recombinant inbred lines of maize. Genome 39:957-968.

AUSTIN, D. F. and M. LEE, 1996b  Comparative mapping in F-2:3 and F-6:7 generations of quantitative trait loci for grain yield and yield components in maize. Theor. Appl. Genet. 92:817-826.

BEAVIS, W. D. and D. GRANT, 1991  A linkage map based on information from four F₂ populations of maize (Zea mays L.). Theor. Appl. Genet. 82:636-644.

BEAVIS, W. D., M. LEE, D. GRANT, A. R. HALLAUER, and T. OWENS et al., 1992  The influence of random mating on recombination among RFLP loci. Maize Gen. Coop. Newsl. 66:52-53.

BECKETT, J. B., 1991 Cytogenetic, genetic, and plant breeding applications of B-A translocations in maize, pp. 493–529 in Chromosome Engineering in Plants: Genetics, Breeding, and Evolution, Part A, edited by P. K. GUPTA and T. TSUCHIYA. Elsevier Science Publishers, Amsterdam.

BENNETZEN, J. L. and M. FREELING, 1993  Grasses as a single genetic system: genome composition, colinearity, and compatibility. Trends Genet. 9:259-261[Medline].

BIRNBOIM, H. C., 1983  A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 100:243-255[Medline].

BOGUSKI, M. S., T. M. LOWE, and C. M. TOLSTOSHEV, 1993  dbEST—database for "expressed sequence tags". Nat. Genet. 4(4):332-333[Medline].

BOHN, M., M. KHAIRALLAH, D. GONZALEZ DE LEON, D. A. HOISINGTON, and H. UTZ et al., 1996  QTL mapping in tropical maize. 1. Genomic regions affecting leaf feeding resistance to sugarcane borer and other traits. Crop Sci. 36:1352-1361[Abstract/Free Full Text].

BURR, B., F. A. BURR, K. H. THOMPSON, M. C. ALBERTSEN, and C. W. STUBER, 1988  Gene mapping with recombinant inbreds in maize. Genetics 118:519-526[Abstract/Free Full Text].

BURR, B., F. A. BURR and E. MATZ, 1996 Molecular map of T232 x CM37 and CO159 x Tx303 recombinant inbred populations. Maize Genome Database (http://www.agron.missouri.edu).

BYRNE, P. F., M. D. MCMULLEN, M. E. SNOOK, T. MUSKET, and J. M. THEURI et al., 1996  Quantitative trait loci and metabolic pathways: genetic control of the concentration of maysin, a corn earworm resistance factor, in maize silks. Proc. Natl. Acad. Sci. USA 93:8820-8825[Abstract/Free Full Text].

CAUSSE, M., J. P. ROCHER, A. M. HENRY, A. CHARCOSSET, and J. L. PRIOUL et al., 1995  Genetic dissection of the relationship between carbon metabolism and early growth in maize, with emphasis on key-enzyme loci. Mol. Breed. 1:259-272.

CAUSSE, M., S. SANTONI, C. DAMERVAL, A. MAURICE, and A. CHARCOSSET et al., 1996  A composite map of expressed sequences in maize. Genome 39:418-432.

CHAO, S., C. BAYSDORFER, O. HEREDIA-DIAZ, T. MUSKET, and G. XU et al., 1994  RFLP mapping of partially sequenced leaf cDNA clones in maize. Theor. Appl. Genet. 88:717-721.

CIMMYT, 1994 QTL data for populations Ki3 x CML139 and CML131 x CML67. Maize Genome Database (http://www.agron.missouri.edu).

COE, E. H., D. A. HOISINGTON, and M. G. NEUFFER, 1987  Linkage map of corn(maize) (Zea mays L.). Maize Genet. Coop. Newslett. 61:116-147.

COE, E. H., G. L. DAVIS, M. D. MCMULLEN, T. MUSKET, and M. POLACCO, 1995  Gene list and working maps. Maize Genet. Coop. Newslett. 69:191-192.

COX, D. R., M. BURMEISTER, E. R. PRICE, S. KIM, and R. M. MYERS, 1990  Radiation hybrid mapping: a somatic cell genetic method for constructing high-resolution maps of mammalian chromosomes. Science 250:245-250[Abstract/Free Full Text].

DAMERVAL, C., A. MAURICE, J. M. JOSSE, and D. DE VIENNE, 1994  Quantitative trait loci underlying gene product variation: a novel perspective for analyzing regulation of genome expression. Genetics 137:289-301[Abstract].

DELSENY, M., R. COOKE, M. RAYNAL, and F. GRELLET, 1997  The Arabidopsis thaliana cDNA sequencing projects. FEBS Letters 405:129-132[Medline].

EDWARDS, M. D., T. HELENTJARIS, S. WRIGHT, and C. W. STUBER, 1992  Molecular-marker-facilitated investigations of quantitative trait loci in maize. 4. Analysis based on genome saturation with isozyme and restriction fragment length polymorphism markers. Theor. Appl. Genet. 83:765-774.

EMERSON, R. A., G. W. BEADLE, and A. C. FRASER, 1935  A summary of linkage studies in maize. Cornell Univ. Agric. Exp. Stn. Memoir 180:1-83.

FROVA, C. and M. SARI-GORLA, 1994  Quantitative trait loci (QTLs) for pollen thermotolerance detected in maize. Mol. Gen. Genet. 245:424-430[Medline].

GARDINER, J., E. H. COE, JR., S. MELIA-HANCOCK, D. A. HOISINGTON, and S. CHAO, 1993  Development of a core RFLP map in maize using an Immortalized-F₂ population. Genetics 134:917-930[Abstract].

HARUSHIMA, Y., M. YANO, A. SHOMURA, M. SATO, and T. SHIMANO et al., 1998  A high-density rice genetic linkage map with 2275 markers using a single F₂ population. Genetics 148:479-494[Abstract/Free Full Text].

HELENTJARIS, T., M. SLOCUM, S. WRIGHT, A. SCHAEFER, and J. NIENHUIS, 1986  Construction of genetic linkage maps in maize and tomato using restriction fragment length polymorphisms. Theor. Appl. Genet. 72:761-769.

HELENTJARIS, T., D. WEBER, and S. WRIGHT, 1988  Identification of the genomic locations of duplicate nucleotide sequences in maize by analysis of restriction fragment length polymorphisms. Genetics 118:353-363[Abstract/Free Full Text].

HULBERT, S. H., T. E. RICHTER, J. D. AXTELL, and J. L. BENNETZEN, 1990  Genetic mapping and characterization of sorghum and related crops by means of maize DNA probes. Proc. Natl. Acad. Sci. USA 87:4251-4255[Abstract/Free Full Text].

KEITH, C. S., D. O. HOANG, B. M. BARRETT, B. FEIGLEMAN, and M. C. NELSON et al., 1993  Partial sequence analysis of 130 randomly selected maize cDNA clones. Plant Physiol. 101:329-332[Abstract].

KURATA, N., Y. NAGAMURA, K. YAMAMOTO, Y. HARUSHIMA, and N. SUE et al., 1994  A 300 kilobase interval genetic map of rice including 883 expressed sequences. Nature Genet. 8:365-372[Medline].

LEBRETON, C., V. LAZIC-JANCIC, A. STEED, S. PEKIC, and S. A. QUARRIE, 1995  Identification of QTL for drought responses in maize and their use in testing causal relationships between traits. J. Exp. Bot. 46:853-865[Abstract/Free Full Text].

LUBBERSTEDT, T., A. E. MELCHINGER, C. C. SCHON, H. UTZ, and D. KLEIN, 1997  QTL mapping in testcrosses of European flint lines of maize. 1. Comparison of different testers for forage yield traits. Crop Sci. 37:921-931.

MATZ, E. C., F. A. BURR, and B. BURR, 1995  Molecular map based on TxCM and COxTx recombinant inbred families. Maize Gen. Coop. Newsl. 69:257-267.

MOORE, G., M. D. GALE, N. KURATA, and R. B. FLAVELL, 1993  Molecular analysis of small grain cereal genomes: current status and prospects. Bio/Technology 11:594-599.

NAGAMURA, Y., B. A. ANTONIO, and T. SASAKI, 1997  Rice molecular genetic map using RFLPs and its application. Plant Mol. Biol. 35:79-87[Medline].

NELSON, S. F., J. H. MCCUSKER, M. A. SANDER, Y. KEE, and P. MODRICH et al., 1993  Genomic mismatch scanning: a new approach to genetic linkage mapping. Nat. Genet. 4(1):11-18[Medline].

PE, M. E., L. GIANFRANCESCHI, G. TARAMINO, R. TARCHINI, and P. ANGELINI et al., 1993  Mapping quantitative trait loci (QTLs) for resistance to Gibberella zeae infection in maize. Mol. Gen. Genet. 241:11-16[Medline].

PHINNEY, B. O., 1984 Gibberellin A1, dwarfism, and the control of shoot elongation in higher plants, pp. 17–41 in The Biosynthesis and Metabolism of Plant Hormones, edited by A. CROZIER and F. R. HILLMAN. Cambridge University Press, Cambridge, United Kingdom.

QUARRIE, S. A., A. STEED, C. LEBRETON, M. GULLI, and C. CALESTANI et al., 1994  QTL analysis of ABA production in wheat and maize and associated physiological traits. Russ. J. Plant Physiol. 41:565-571.

ROMAN, H. and A. J. ULLSTRUP, 1951  The use of A-B translocations to locate genes in maize. Agron. J. 43:450-454[Free Full Text].

SAGHAI-MAROOF, M. A., K. M. SOLIMAN, R. JOGENSEN, and R. W. ALLARD, 1984  Ribosomal DNA Spacer length in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc. Natl. Acad. Sci. USA 81:8014-8018[Abstract/Free Full Text].

SARI-GORLA, M., M. E. PE, and L. ROSSINI, 1994  Detection of QTLs controlling pollen germination and growth in maize. Heredity 72:332-335.

SCHMIDT, R., J. WEST, K. LOVE, Z. LENCHAN, and D. LISTER et al., 1995  Physical map and organization of Arabidopsis thaliana chromosome 4. Science 270:480-483[Abstract/Free Full Text].

SCHULER, G. D., M. S. BOGUSKI, E. A. STEWART, L. D. STEIN, and G. GYAPAY et al., 1996  A gene map of the human genome. Science 274:540-546[Abstract/Free Full Text].

SENIOR, M. L. and M. HEUN, 1993  Mapping maize microsatellites and polymerase chain reaction confirmation of the targeted repeats using a CT primer. Genome 36(5):884-889[Medline].

SHALON, D., S. J. SMITH, and P. O. BROWN, 1996  A DNA micro-array system for analyzing complex DNA samples using two-color fluorescent probe hybridization. Genome Res. 6:639-645[Abstract/Free Full Text].

STEWART, E. A., K. B. MCKUSICK, A. AGGARWAL, E. BAJOREK, and S. BRADY et al., 1997  An STS-based radiation hybrid map of the human genome. Genome Res. 7(5):422-433[Abstract/Free Full Text].

TARAMINO, G. and S. TINGEY, 1996  Simple sequence repeats for germplasm analysis and mapping in maize. Genome 39(2):277-287[Medline].

VELDBOOM, L. R. and M. LEE, 1994  Molecular-marker-facilitated studies of morphological traits in maize. 2. Determination of QTLs for grain yield and yield components. Theor. Appl. Genet. 89:451-458.

VELDBOOM, L. R., M. LEE, and W. L. WOODMAN, 1994  Molecular-marker-facilitated studies in an elite maize population. 1. Linkage analysis and determination of QTL for morphological traits. Theor. Appl. Genet. 88:7-16.

WEBER, D. and T. HELENTJARIS, 1989  Mapping RFLP loci in maize using B-A translocations. Genetics 121:583-590[Abstract/Free Full Text].

WENDEL, J. F., C. W. STUBER, M. D. EDWARDS, and M. M. GOODMAN, 1986  Duplicated chromosome segments in maize (Zea mays L.): further evidence from hexokinase isozymes. Theor. Appl. Genet. 72:178-185.

WHITKUS, R., J. DOEBLEY, and M. LEE, 1992  Comparative genome mapping of sorghum and maize. Genetics 132:1119-1130[Abstract].

YAMAMOTO, K. and T. SASAKI, 1997  Large-scale EST sequencing in rice. Plant Mol. Biol. 35:135-144[Medline].

ZACHGO, E. A., M. L. WANG, J. DEWDNEY, D. BOUCHEZ, and C. CAMILLERI et al., 1996  A physical map of chromosome 2 of Arabidopsis thaliana.. Genome Res. 6:19-25[Abstract/Free Full Text].

ZHANG, H. B. and R. A. WING, 1997  Physical mapping of the rice genome with BACs. Plant Mol. Biol. 35:115-127[Medline].

This article has been cited by other articles:

				F. I. E. Amarillo and H. W. Bass A Transgenomic Cytogenetic Sorghum (Sorghum propinquum) Bacterial Artificial Chromosome Fluorescence in Situ Hybridization Map of Maize (Zea mays L.) Pachytene Chromosome 9, Evidence for Regions of Genome Hyperexpansion Genetics, November 1, 2007; 177(3): 1509 - 1526. [Abstract] [Full Text] [PDF]

				S.-B. Chang, L. K. Anderson, J. D. Sherman, S. M. Royer, and S. M. Stack Predicting and Testing Physical Locations of Genetically Mapped Loci on Tomato Pachytene Chromosome 1 Genetics, August 1, 2007; 176(4): 2131 - 2138. [Abstract] [Full Text] [PDF]

				T. Nakazato, M.-K. Jung, E. A. Housworth, L. H. Rieseberg, and G. J. Gastony Genetic Map-Based Analysis of Genome Structure in the Homosporous Fern Ceratopteris richardii Genetics, July 1, 2006; 173(3): 1585 - 1597. [Abstract] [Full Text] [PDF]

				L. A. Robertson-Hoyt, M. P. Jines, P. J. Balint-Kurti, C. E. Kleinschmidt, D. G. White, G. A. Payne, C. M. Maragos, T. L. Molnar, and J. B. Holland QTL Mapping for Fusarium Ear Rot and Fumonisin Contamination Resistance in Two Maize Populations Crop Sci., June 20, 2006; 46(4): 1734 - 1743. [Abstract] [Full Text] [PDF]

				C.-J. R. Wang, L. Harper, and W. Z. Cande High-Resolution Single-Copy Gene Fluorescence in Situ Hybridization and Its Use in the Construction of a Cytogenetic Map of Maize Chromosome 9 PLANT CELL, March 1, 2006; 18(3): 529 - 544. [Abstract] [Full Text] [PDF]

				C. J. Lawrence, T. E. Seigfried, H. W. Bass, and L. K. Anderson Predicting Chromosomal Locations of Genetically Mapped Loci in Maize Using the Morgan2McClintock Translator Genetics, March 1, 2006; 172(3): 2007 - 2009. [Abstract] [Full Text] [PDF]

				L. K. Anderson, A. Lai, S. M. Stack, C. Rizzon, and B. S. Gaut Uneven distribution of expressed sequence tag loci on maize pachytene chromosomes Genome Res., January 1, 2006; 16(1): 115 - 122. [Abstract] [Full Text] [PDF]

				I. Vroh Bi, M. D. McMullen, H. Sanchez-Villeda, S. Schroeder, J. Gardiner, M. Polacco, C. Soderlund, R. Wing, Z. Fang, and E. H. Coe Jr. Single Nucleotide Polymorphisms and Insertion-Deletions for Genetic Markers and Anchoring the Maize Fingerprint Contig Physical Map Crop Sci., December 2, 2005; 46(1): 12 - 21. [Abstract] [Full Text] [PDF]