Agronomy

Sugarbeet cultivars are almost exclusively hybrids, which are produced using the sole source of cytoplasmic male sterility (CMS), the so-called Owen CMS. Several alternative sources of CMS have been described. One of these, I-12CMS(3), was derived from wild beets collected in Pakistan, and another CMS source, GCMS, has a cytoplasmic origin in wild sea beets from France. During the past decade, male sterility-associated mitochondrial genes have been identified in these three CMS systems. Moreover, the recent development of a variety of DNA markers has permitted the genetic mapping of nuclear restorer-of-fertility genes for both Owen and GCMS. This review focuses on the mechanism of CMS in beets.

Laser-capture microdissection (LCM) allows for the one-step procurement of large homogeneous populations of cells from tissue sections. In mammals, LCM has been used to conduct cDNA microarray and proteomics studies on specific cell types. However, LCM has not been applied to plant cells, most likely because plant cell walls make it difficult to separate target cells from surrounding cells and because ice crystals can form in the air spaces between cells when preparing frozen sections. By fixing tissues, using a cryoprotectant before freezing, and using an adhesive-coated slide system, it was possible to capture large numbers ( Ͼ 10,000) of epidermal cells and vascular tissues (vascular bundles and bundle sheath cells) from ethanol:acetic acid-fixed coleoptiles of maize. RNA extracted from these cells was amplified with T7 RNA polymerase and used to hybridize a microarray containing ‫ف‬ 8800 maize cDNAs. Approximately 250 of these were expressed preferentially in epidermal cells or vascular tissues. These results demonstrate that the combination of LCM and microarrays makes it feasible to conduct high-resolution global gene expression analyses of plants. This approach has the potential to enhance our understanding of diverse plant cell type-specific biological processes.

Some plant cytoplasms express novel mitochondrial genes that cause male sterility. Nuclear genes that disrupt the accumulation of the corresponding mitochondrial gene products can restore fertility to such plants. The Texas (T) cytoplasm mitochondrial genome of maize expresses a novel protein, URF13, which is necessary for T cytoplasm-induced male sterility. Working in concert, functional alleles of two nuclear genes, rf1 and rf2 , can restore fertility to T cytoplasm plants. Rf1 alleles, but not Rf2 alleles, reduce the accumulation of URF13. Hence, Rf2 differs from typical nuclear restorers in that it does not alter the accumulation of the mitochondrial protein necessary for T cytoplasm-induced male sterility. This study established that the rf2 gene encodes a soluble protein that accumulates in the mitochondrial matrix. Three independent lines of evidence establish that the RF2 protein is an aldehyde dehydrogenase (ALDH). The finding that T cytoplasm plants that are homozygous for the rf2-R213 allele are male sterile but accumulate normal amounts of RF2 protein that lacks normal mitochondrial (mt) ALDH activity provides strong evidence that rf2 -encoded mtALDH activity is required to restore male fertility to T cytoplasm maize. Detailed genetic analyses have established that the rf2 gene also is required for anther development in normal cytoplasm maize. Hence, it appears that the rf2 gene was recruited recently to function as a nuclear restorer. ALDHs typically have very broad substrate specificities. Indeed, the RF2 protein is capable of oxidizing at least three aldehydes. Hence, the specific metabolic pathway(s) within which the rf2 -encoded mtALDH acts remains to be discovered. Yoder, O.C. (1973). A selective toxin produced by Phyllosticta maydis. Phytopathology 63, 1361-1366. Yoshida, A., Rzhetsky, A., Hsu, L.C., and Chang, C. (1998). Human aldehyde dehydrogenase gene family. Eur. J. Biochem. 251, 549-557. Zinovieva, R.D., Tomarev, S.I., and Piatigorsky, J. (1993). Aldehyde dehydrogenase-derived omega-crystallins of squid and octopus: Specialization for lens expression.

A massively parallel pyro-sequencing technology commercialized by 454 Life Sciences Corporation was used to sequence the transcriptomes of shoot apical meristems isolated from two inbred lines of maize using laser capture microdissection (LCM). A computational pipeline that uses the POLYBAYES polymorphism detection system was adapted for 454 ESTs and used to detect SNPs (single nucleotide polymorphisms) between the two inbred lines. Putative SNPs were computationally identified using 260 000 and 280 000 454 ESTs from the B73 and Mo17 inbred lines, respectively. Over 36 000 putative SNPs were detected within 9980 unique B73 genomic anchor sequences (MAGIs). Stringent post-processing reduced this number to > 7000 putative SNPs. Over 85% (94/110) of a sample of these putative SNPs were successfully validated by Sanger sequencing. Based on this validation rate, this pilot experiment conservatively identified > 4900 valid SNPs within > 2400 maize genes. These results demonstrate that 454-based transcriptome sequencing is an excellent method for the high-throughput acquisition of gene-associated SNPs.

Heterosis is the phenomenon whereby the progeny of particular inbred lines have enhanced agronomic performance relative to both parents. Although several hypotheses have been proposed to explain this fundamental biological phenomenon, the responsible molecular mechanisms have not been determined. The maize inbred lines B73 and Mo17 produce a heterotic F1 hybrid. Global patterns of gene expression were compared in seedlings of these three genotypes by using a microarray that contains 13,999 cDNAs. Using an estimated 15% false discovery rate as a cutoff, 1,367 ESTs (9.8%) were identified as being significantly differentially expressed among genotypes. All possible modes of gene action were observed, including additivity, high-and low-parent dominance, underdominance, and overdominance. The largest proportion of the ESTs (78%; 1,062 of 1,367) exhibited expression patterns that are not statistically distinguishable from additivity. Even so, 22% of the differentially regulated ESTs exhibited nonadditive modes of gene expression. Classified on the basis of significant pairwise comparisons of genotype means, 181 of these 305 nonadditive ESTs exhibited high-parent dominance and 23 exhibited lowparent dominance. In addition, 44 ESTs exhibited underdominance or overdominance. These findings are consistent with the hypothesis that multiple molecular mechanisms, including overdominance, contribute to heterosis. global transcript profiling ͉ heterosis ͉ overdominance

A 470-kb segment from the long arm of chromosome 3 of Zea mays (inbred LH82), encompassing the al-sh2 interval, was cloned as a yeast artificial chromosome. Comparison of the sizes of the restriction fragments generated from the cloned DNA fragment and from the DNA isolated from the maize inbred line LH82 established the colinearity of the al-sh2 interval in these DNAs. By utilizing a chromosome fragmentation technique, a yeast artificial chromosome encompassing the al-sh2 interval was separately fragmented at the a] and sh2 loci. Comparison of the sizes of these fragmentation products established the physical distance between the al and sh2 loci to be 140 kb. Furthermore, these fragmentation experiments established the physical orientation of the a) and sh2 genes relative to the maize centromere. The molecular cloning of the contiguous region between the a) and sh2 loci made it possible to define the relationship between physical and genetic distances over a relatively large segment of the maize genome. In this interval, the relationship between physical and genetic distances is 1560 kb/centimorgan, which compares with 1460 kb/centimorgan for the entire maize genome, and 217 kb/centimorgan for a 1-kb segment within the al locus.

Here we present a routine and efficient protocol for year-round production of fertile transgenic maize plants. Type II callus derived from maize Hi II immature zygotic embryos was transformed using the PDS 1000/He biolistic gun and selected on bialaphos. In an effort to improve the transformation protocol, the effects of gold particle size and callus morphology on transformation efficiency were investigated. Reducing gold particle size from 1.0 mm or 0.6 mm resulted in a significant increase in the rate of recovery of bialaphos-resistant clones from Type II callus. The average transformation efficiency of pre-embryogenic, early embryogenic and late embryogenic callus did not vary significantly. Rates of transformation, regeneration and fertility achieved for Type II callus are summarized and compared to those achieved for greenhouse-and field-derived immature zygotic embryos.

Many large genomes are getting sequenced nowadays. Biologists are eager to start microarray analysis taking advantage of all known genes of a species, but existing microarray design tools were very inefficient for large genomes. Also, many existing tools operate in a batch mode that does not assure best designs.

Recent sequencing efforts have targeted the gene-rich regions of the maize (Zea mays L.) genome. We report the release of an improved assembly of maize assembled genomic islands (MAGIs). The 114,173 resulting contigs have been subjected to computational and physical quality assessments. Comparisons to the sequences of maize bacterial artificial chromosomes suggest that at least 97% (160 of 165) of MAGIs are correctly assembled. Because the rates at which junction-testing PCR primers for genomic survey sequences (90 -92%) amplify genomic DNA are not significantly different from those of control primers (Ϸ91%), we conclude that a very high percentage of genic MAGIs accurately reflect the structure of the maize genome. EST alignments, ab initio gene prediction, and sequence similarity searches of the MAGIs are available at the Iowa State University MAGI web site. This assembly contains 46,688 ab initio predicted genes. The expression of almost half of a sample of the predicted genes that lack expression evidence was validated by RT-PCR. Our analyses suggest that the maize genome contains between Ϸ33,000 and Ϸ54,000 expressed genes. Approximately 5% (32 of 628) of the maize transcripts discovered do not have detectable paralogs among maize ESTs or detectable homologs from other species in the GenBank NR nucleotide͞protein database. Analyses therefore suggest that this assembly of the maize genome contains approximately 350 previously uncharacterized expressed genes. We hypothesize that these ''orphans'' evolved quickly during maize evolution and͞or domestication.

Plant development is controlled by both endogenous genetic programs and responses to exogenous signals. Microarray experiments are being used to identify the genes involved in these developmental processes. Most of the analyses conducted to date have been conducted on whole organs. Although these studies have provided valuable information, they are limited by the composite nature of plant organs that consist of multiple cell types. Technical advances that have made it possible to study global patterns of gene expression in individual cell types promise to increase greatly the information revealed by microarray experiments.

Because the bulk of the maize (Zea mays L.) genome consists of repetitive sequences, sequencing efforts are being targeted to its 'gene-rich' fraction. Traditional assembly programs are inadequate for this approach because they are optimized for a uniform sampling of the genome and inherently lack the ability to differentiate highly similar paralogs. Results: We report the development of bioinformatics tools for the accurate assembly of the maize genome. This software, which is based on innovative parallel algorithms to ensure scalability, assembled 730 974 genomic survey sequences fragments in 4 h using 64 Pentium III 1.26 GHz processors of a commodity cluster. Algorithmic innovations are used to reduce the number of pairwise alignments significantly without sacrificing quality. Clone pair information was used to estimate the error rate for improved differentiation of polymorphisms versus sequencing errors. The assembly was also used to evaluate the effectiveness of various filtering strategies and thereby provide information that can be used to focus subsequent sequencing efforts.

The 140-kb a1-sh2 interval of the maize genome contains at least four genes (a1, yz1, x1, and sh2). Partial sequence analysis of two haplotypes has revealed many single nucleotide polymorphisms and InDel polymorphisms, including several large structural polymorphisms. The physical positions of 101 meiotic recombination breakpoints are not distributed uniformly across the interval and are instead concentrated within three recombination hot spots. Two of these recombination hot spots are genic (a1 and yz1) and one is apparently nongenic. The x1 gene is not a recombination hot spot. Thus, these results suggest that not all hot spots are genes and indicate that not all genes are hot spots. Two of the 101 recombination events arose by means of either noncrossover events involving conversion tract lengths of at least 17 kb or double-crossover events. Only one recombination breakpoint mapped to the Ϸ80-kb distal portion of the a1-sh2 interval that contains large amounts of repetitive DNA including retrotransposons; in this region the ratio of genetic to physical distance is less than 0.5% of the genome's average. These results establish that the retrotransposon faction of the maize genome is relatively inert recombinationally.

The receptive component of theCy transposable element system (rcy: Mu7) at theBz locus ofZea mays L. is 2.2 kb and has long terminal inverted repeats. The insertion is flanked by a 9 bp duplication. In the presence of an autonomousCy element in the genome,rcy: Mu7 is excised frombz-rcy in a manner consistent with a model suggested previously. The termini ofrcy: Mu7 have 85% sequence similarity with theMu1 element ofZ. mays. This is consistent with the observation thatMu1 can behave genetically like a receptive component of theCy system.

All above-ground plant organs are derived from shoot apical meristems (SAMs). Global analyses of gene expression were conducted on maize (Zea mays L.) SAMs to identify genes preferentially expressed in the SAM. The SAMs were collected from 14-day-old B73 seedlings via laser capture microdissection (LCM). The RNA samples extracted from LCM-collected SAMs and from seedlings were hybridized to microarrays spotted with 37 660 maize cDNAs. Approximately 30% (10 816) of these cDNAs were prepared as part of this study from manually dissected B73 maize apices. Over 5000 expressed sequence tags (ESTs) (about 13% of the total) were differentially expressed (P < 0.0001) between SAMs and seedlings. Of these, 2783 and 2248 ESTs were up-and down-regulated in the SAM, respectively. The expression in the SAM of several of the differentially expressed ESTs was validated via quantitative RT-PCR and/or in situ hybridization. The up-regulated ESTs included many regulatory genes including transcription factors, chromatin remodeling factors and components of the gene-silencing machinery, as well as about 900 genes with unknown functions. Surprisingly, transcripts that hybridized to 62 retrotransposon-related cDNAs were also substantially up-regulated in the SAM. Complementary DNAs derived from the LCM-collected SAMs were sequenced to identify additional genes that are expressed in the SAM. This generated around 550 000 ESTs (454-SAM ESTs) from two genotypes. Consistent with the microarray results, approximately 14% of the 454-SAM ESTs from B73 were retrotransposon-related. Possible roles of genes that are preferentially expressed in the SAM are discussed.

A new genetic map of maize, ISU-IBM Map4, that integrates 2029 existing markers with 1329 new indel polymorphism (IDP) markers has been developed using intermated recombinant inbred lines (IRILs) from the intermated B73 3 Mo17 (IBM) population. The website http:/ /magi.plantgenomics.iastate.edu provides access to IDP primer sequences, sequences from which IDP primers were designed, optimized markerspecific PCR conditions, and polymorphism data for all IDP markers. This new gene-based genetic map will facilitate a wide variety of genetic and genomic research projects, including map-based genome sequencing and gene cloning. The mosaic structures of the genomes of 91 IRILs, an important resource for identifying and mapping QTL and eQTL, were defined. Analyses of segregation data associated with markers genotyped in three B73/Mo17-derived mapping populations (F 2 , Syn5, and IBM) demonstrate that allele frequencies were significantly altered during the development of the IBM IRILs. The observations that two segregation distortion regions overlap with maize flowering-time QTL suggest that the altered allele frequencies were a consequence of inadvertent selection. Detection of two-locus gamete disequilibrium provides another means to extract functional genomic data from well-characterized plant RILs.

The previously cloned CERZ gene is required for the normal accumulation of cuticular waxes and encodes a nove1 protein. Earlier reports suggested that the CERZ protein is either a membrane-bound component of the fatty acid elongase complex or a regulatory protein. Cell fractionation and immunoblot analyses using polyclonal antibodies raised against a chemically synthesized peptide with a sequence based on the predicted CERZ protein sequence have demonstrated that the 47-kD CERZ protein is soluble and nuclear localized. These results are consistent with CERZ being a regulatory pro-

T (Texas) cytoplasm is associated with a mitochondrial disruption that is phenotypically expressed during microsporogenesis resulting in male sterility. Restoration of pollen fertility in T-cytoplasm maize is controlled by dominant alleles at two unlinked, complementary, nuclearencoded genes, rfl and rf2. As a first step in the molecular isolation of the rf2 gene, 178,300 gametes derived from plants that carried the Mutator, Cy or Spm transposon families were screened for rf2 mutant alleles (rf2-m) via their inability to restore pollen fertility to T-cytoplasm male-sterile maize. Seven heritable rf2-m alleles were recovered from these transposon populations. Pedigrees and restriction fragment length polymorphism (RFLP)based analyses indicated that all seven rf2-m alleles were derived independently. The ability to obtain rf2-m derivatives from Rf 2 suggests that Rf 2 alleles produce a functional product necessary to restore pollen fertility to cmsT. Molecular markers flanking the rfl and rf2 loci were used to decipher segregation patterns in progenies segregating for the rf 2-m alleles. These analyses provided preliminary evidence of a weak, third restorer gene of cmsT that can substitute for Rfl.