Mary L. Polacco

Development and mapping of SSR markers for maize.

Microsatellite or simple sequence repeat (SSR) markers have wide applicability for genetic analysis in crop plant improvement strategies. The objectives of this project were to isolate, characterize, and map a comprehensive set of SSR markers for maize (Zea mays L.). We developed 1051 novel SSR markers for maize from microsatellite-enriched libraries and by identification of microsatellite-containing sequences in public and private databases. Three mapping populations were used to derive map positions for 978 of these markers. The main mapping population was the intermated B73 × Mo17 (IBM) population. In mapping this intermated recombinant inbred line population, we have contributed to development of a new high-resolution map resource for maize. The primer sequences, original sequence sources, data on polymorphisms across 11 inbred lines, and map positions have been integrated with information on other public SSR markers and released through MaizeDB at URL:www.agron.missouri.edu. The maize research community now has the most detailed and comprehensive SSR marker set of any plant species.

Anchoring 9,371 Maize Expressed Sequence Tagged Unigenes to the Bacterial Artificial Chromosome Contig Map by Two-Dimensional Overgo Hybridization

Our goal is to construct a robust physical map for maize (Zea mays) comprehensively integrated with the genetic map. We have used a two-dimensional 24 × 24 overgo pooling strategy to anchor maize expressed sequence tagged (EST) unigenes to 165,888 bacterial artificial chromosomes (BACs) on high-density filters. A set of 70,716 public maize ESTs seeded derivation of 10,723 EST unigene assemblies. From these assemblies, 10,642 overgo sequences of 40 bp were applied as hybridization probes. BAC addresses were obtained for 9,371 overgo probes, representing an 88% success rate. More than 96% of the successful overgo probes identified two or more BACs, while 5% identified more than 50 BACs. The majority of BACs identified (79%) were hybridized with one or two overgos. A small number of BACs hybridized with eight or more overgos, suggesting that these BACs must be gene rich. Approximately 5,670 overgos identified BACs assembled within one contig, indicating that these probes are highly locus specific. A total of 1,795 megabases (Mb; 87%) of the total 2,050 Mb in BAC contigs were associated with one or more overgos, which are serving as sequence-tagged sites for single nucleotide polymorphism development. Overgo density ranged from less than one overgo per megabase to greater than 20 overgos per megabase. The majority of contigs (52%) hit by overgos contained three to nine overgos per megabase. Analysis of approximately 1,022 Mb of genetically anchored BAC contigs indicates that 9,003 of the total 13,900 overgo-contig sites are genetically anchored. Our results indicate overgos are a powerful approach for generating gene-specific hybridization probes that are facilitating the assembly of an integrated genetic and physical map for maize.

Genetic, Physical, and Informatics Resources for Maize. On the Road to an Integrated Map

Maize ( Zea mays ) is among the most important crop plants in the world. For any crop plant, an integrated genetic and physical map serves as the foundation for numerous studies, especially those aimed at improving the agronomic characteristics of the plant. Once a phenotypically defined locus

Characterization of Three Maize Bacterial Artificial Chromosome Libraries toward Anchoring of the Physical Map to the Genetic Map Using High-Density Bacterial Artificial Chromosome Filter Hybridization

Three maize (Zea mays) bacterial artificial chromosome (BAC) libraries were constructed from inbred line B73. High-density filter sets from all three libraries, made using different restriction enzymes (HindIII,EcoRI, and MboI, respectively), were evaluated with a set of complex probes including the185-bp knob repeat, ribosomal DNA, two telomere-associated repeat sequences, four centromere repeats, the mitochondrial genome, a multifragment chloroplast DNA probe, and bacteriophage λ. The results indicate that the libraries are of high quality with low contamination by organellar and λ-sequences. The use of libraries from multiple enzymes increased the chance of recovering each region of the genome. Ninety maize restriction fragment-length polymorphism core markers were hybridized to filters of the HindIII library, representing 6× coverage of the genome, to initiate development of a framework for anchoring BAC contigs to the intermated B73 × Mo17 genetic map and to mark the bin boundaries on the physical map. All of the clones used as hybridization probes detected at least three BACs. Twenty-two single-copy number core markers identified an average of 7.4 ± 3.3 positive clones, consistent with the expectation of six clones. This information is integrated into fingerprinting data generated by the Arizona Genomics Institute to assemble the BAC contigs using fingerprint contig and contributed to the process of physical map construction.

Aberrations in plastid transcripts and deficiency of plastid DNA in striped and albino mutants in maize

To better understand the regulatory roles of nuclear genes in chloroplast genomic expression during leaf development in maize (Zea mays L.), we studied a striped mutant, ii1 (iojap 1), two albino mutants, w1 (white 1) and w2 (white 2), and their double mutants with l (luteus). Homozygous ij1 plants as a female parent produce albino seedlings, called maternal exceptions, among their progeny, even when the nuclear genotype of the male parent is normal (+/+). In contrast to albinos that are blocked in the biosynthetic pathway of carotenoids, w1 and w2 seedlings fail to accumulate chlorophyll and carotenoids up to the normal level even under dim light conditions. In ij1-affected plastids, the plastid-encoded proteins and nuclear-encoded proteins that are associated with thylakoid membranes were not detecable. However, the 33-kDa protein of the oxygen-evolving complex and ferredoxin: NADP oxidoreductase, which are localized extrinsically, were accumulated even though the level of the proteins was decreased. Both ij1 and w1 albino seedlings contain a normal level of plastid DNA. However, both show similar aberrant patterns among the transcripts of all the plastid genes examined (psbB, psbH, petB, petD, atpA, psaB, psbA, and rbcL). Not only were additional transcripts detected but some of the normal transcripts were not detectable or were barely detectable by Northern hybridization. These facts indicate that the transcripts of ij1- and w1-affected plastids may have altered synthesis, processing or stability. Therefore, the block in expression of the plastid genome by the nuclear mutants ij1 and w1 may be due to alterations in the transcriptional or post-transcriptional processes. The fact that ij1 and maternal-exception progeny show almost identical patterns of transcripts indicates that the effects of ij1 on plastid gene expression persist in the subsequent generation even after the nuclear gene, Ij1, restores the normal function. In contrast to ij1 and w1, the levels of all plastid transcripts in w2 seedlings, whether l or +, are uniformly reduced. Compared to normal sibling seedlings, the patterns of the RNA species are relatively unaltered. Relative to the level of a nuclear rDNA, the plastid DNA content of w2 is decreased 20-fold. Therefore, the limited expression of the w2-affected plastids may be due to failure to maintain the copy number of plastid genomes. Thus, albinisms of these mutants result from limiting of expression of plastids due to alteration of transcripts on the one hand, or to lowered DNA content on the other.

Development of an integrated laboratory information management system for the maize mapping project

MOTIVATION The development of an integrated genetic and physical map for the maize genome involves the generation of an enormous amount of data. Managing this data requires a system to aid in genotype scoring for different types of markers coming from both local and remote users. In addition, researchers need an efficient way to interact with genetic mapping software and with data files from automated DNA sequencing. They also need ways to manage primer data for mapping and sequencing and provide views of the integrated physical and genetic map and views of genetic map comparisons. RESULTS The MMP-LIMS system has been used successfully in a high-throughput mapping environment. The genotypes from 957 SSR, 1023 RFLP, 189 SNP, and 177 InDel markers have been entered and verified via MMP-LIMS. The system is flexible, and can be easily modified to manage data for other species. The software is freely available. AVAILABILITY To receive a copy of the iMap or cMap software, please fill out the form on our website. The other MMP-LIMS software is freely available at http://www.maizemap.org/bioinformatics.htm.

Mini-preparation of highly purified chloroplast DNA from maize

W E have an interest in isolating chloroplast DNA (cpDNA) from maternally inherited maize photosynthesis mutants where tissue availability is limited and have devised a procedure that gives high yields of pure cpDNA from maize seedlings. Highly purified cpDNA requires the use of DNAase to remove contaminating nuclear DNA from intact chloroplast preparations (Kolodner and Tewari, 1975; Herrmann et al., 1975; Bogorad et al., 1983). The DNAase is typically inactivated by pronase prior to rupture of chloroplasts to release cpDN A. Commercial pronase contains low levels of nuclease activities, which are removed by self-digestion. In maize, yields of 100-200 ~tg cpDNA are obtained per kg seedling leaves (Bogorad et al., 1983). We describe a procedure that gives improved yields by incorporating two modifications of established protocols: a three-day rather than a one-day etiolation period prior to isolation of chloroplasts and substi tu tion of nucleasefree proteinase K for pronase. Starting with 10 grn of material, we routinely obtain 10-15 ~g cpDNA that is free of nuclear DNA.

A maternally inherited mutant of Zea mays L. lacks the cytochrome b/f complex

SummaryA maternally inherited yellowgreen mutant (myg-1) of maize is described that lacks the thylakoid cytochrome b/f complex. Affected individuals segregate in sectors on ears indicating that a cytoplasmic genetic component is affected. Because there is an effect on a thylakoid complex, and completely yellow-green mutant seedlings grow normally until endosperm reserves are exhausted, the lesion is probably in the plastome and not in the mitochondrial genome. Southern blot analyses show no major insertions or deletions in or surrounding plastome genes pet A and pet B, which encode subunits of the missing cytochrome complex. There is, however, a significant decrease in transcripts of these genes. The myg-1 mutation is the first non-Mendelian maize mutant which has been reported to affect a defined chloroplast complex.

A nuclear encoded chloroplast ATP synthase mutant of Zea mays L

SummaryNumerous Escherichia coli mutants have been used to determine the genetics and sequence of assembly of the prokaryotic proton-translocating ATP synthase complex. Similar studies with the analogous chloroplast ATP synthase in higher plants have not been possible due to lack of suitable mutants. We describe here a preliminary characterization of cfr, a nuclear mutation in Zea mays L. that appears to destabilize or prevent assembly of the chloroplast ATP synthase complex. Biochemical and physiological analyses indicate that the amounts of both the CF1 and CFo components of the complex are severely diminished. Mutant seedlings are pale green and occasionally survive, with greatly reduced vigor, to maturity. The cfr locus has been mapped genetically to the short arm of chromosome. 1.

MaizeDB – A Functional Genomics Perspective

MaizeDB (http://www.agron.missouri.edu/) has existed since the early 90’s as a genomespecific database that is grounded in genetic maps, their documentation and annotation. The database management system is robust and has continuously been Sybase. In this brief review we provide an introduction to the database as a functional genomics tool and new accesses to the data: 1) probe tables by bin location 2) BLAST access to map data 3) cMap, a comparative map graphical tool.