A Critical Assessment of 60 Years of Maize Intragenic Recombination.

Until the mid-1950s, it was believed that genetic crossovers did not occur within genes. Crossovers occurred between genes, the "beads on a string" model. Then in 1956, Seymour Benzer published his classic paper describing crossing over within a gene, intragenic recombination. This result from a bacteriophage gene prompted Oliver Nelson to study intragenic recombination in the maize Waxy locus. His studies along with subsequent work by others working with maize and other organisms described the outcomes of intragenic recombination and provided some of the earliest evidence that genes, not intergenic regions, were recombination hotspots. High-throughput genotyping approaches have since replaced single gene intragenic studies for characterizing the outcomes of recombination. These large-scale studies confirm that genes, or more generally genic regions, are the most active recombinogenic regions, and suggested a pattern of crossovers similar to the budding yeast Saccharomyces cerevisiae. In S. cerevisiae recombination is initiated by double-strand breaks (DSBs) near transcription start sites (TSSs) of genes producing a polarity gradient where crossovers preferentially resolve at the 5' end of genes. Intragenic studies in maize yielded less evidence for either polarity or for DSBs near TSSs initiating recombination and in certain respects resembled Schizosaccharomyces pombe or mouse. These different perspectives highlight the need to draw upon the strengths of different approaches and caution against relying on a single model system or approach for understanding recombination.

ELIGULUM-A Regulates Lateral Branch and Leaf Development in Barley.

The shoot apical and axillary meristems control shoot development, effectively influencing lateral branch and leaf formation. The barley (Hordeum vulgare) uniculm2 (cul2) mutation blocks axillary meristem development, and mutant plants lack lateral branches (tillers) that normally develop from the crown. A genetic screen for cul2 suppressors recovered two recessive alleles of ELIGULUM-A (ELI-A) that partially rescued the cul2 tillering phenotype. Mutations in ELI-A produce shorter plants with fewer tillers and disrupt the leaf blade-sheath boundary, producing liguleless leaves and reduced secondary cell wall development in stems and leaves. ELI-A is predicted to encode an unannotated protein containing an RNaseH-like domain that is conserved in land plants. ELI-A transcripts accumulate at the preligule boundary, the developing ligule, leaf margins, cells destined to develop secondary cell walls, and cells surrounding leaf vascular bundles. Recent studies have identified regulatory similarities between boundary development in leaves and lateral organs. Interestingly, we observed ELI-A transcripts at the preligule boundary, suggesting that ELI-A contributes to boundary formation between the blade and sheath. However, we did not observe ELI-A transcripts at the axillary meristem boundary in leaf axils, suggesting that ELI-A is not involved in boundary development for axillary meristem development. Our results show that ELI-A contributes to leaf and lateral branch development by acting as a boundary gene during ligule development but not during lateral branch development.

The barley Uniculme4 gene encodes a BLADE-ON-PETIOLE-like protein that controls tillering and leaf patterning.

Tillers are vegetative branches that develop from axillary buds located in the leaf axils at the base of many grasses. Genetic manipulation of tillering is a major objective in breeding for improved cereal yields and competition with weeds. Despite this, very little is known about the molecular genetic bases of tiller development in important Triticeae crops such as barley (Hordeum vulgare) and wheat (Triticum aestivum). Recessive mutations at the barley Uniculme4 (Cul4) locus cause reduced tillering, deregulation of the number of axillary buds in an axil, and alterations in leaf proximal-distal patterning. We isolated the Cul4 gene by positional cloning and showed that it encodes a BROAD-COMPLEX, TRAMTRACK, BRIC-À-BRAC-ankyrin protein closely related to Arabidopsis (Arabidopsis thaliana) BLADE-ON-PETIOLE1 (BOP1) and BOP2. Morphological, histological, and in situ RNA expression analyses indicate that Cul4 acts at axil and leaf boundary regions to control axillary bud differentiation as well as the development of the ligule, which separates the distal blade and proximal sheath of the leaf. As, to our knowledge, the first functionally characterized BOP gene in monocots, Cul4 suggests the partial conservation of BOP gene function between dicots and monocots, while phylogenetic analyses highlight distinct evolutionary patterns in the two lineages.

A Simple Method for the Assessment of Fusarium Head Blight Resistance in Korean Wheat Seedlings Inoculated with Fusarium graminearum.

Fusarium head blight (FHB; scab) caused mainly by Fusarium graminearum is a devastating disease of wheat and barley around the world. FHB causes yield reductions and contamination of grain with trichothecene mycotoxins such as deoxynivalenol (DON) which are a major health concern for humans and animals. The objective of this research was to develop an easy seed or seedling inoculation assay, and to compare these assays with whole plant resistance of twenty-nine Korean winter wheat cultivars to FHB. The clip-dipping assay consists of cutting off the coleoptiles apex, dipping the coleoptiles apex in conidial suspension, covering in plastic bag for 3 days, and measuring the lengths of lesions 7 days after inoculation. There were significant cultivar differences after inoculation with F. graminearum in seedling relative to the controls. Correlation coefficients between the lesion lengths of clip-dipping inoculation and FHB Type II resistance from adult plants were significant (r=0.45; P<0.05). Results from two other seedling inoculation methods, spraying and pin-point inoculation, were not correlated with adult FHB resistance. Single linear correlation was not significant between seed germination assays (soaking and soak-dry) and FHB resistance (Type I and Type II), respectively. These results showed that clip-dipping inoculation method using F. graminearum may offer a real possibility of simple, rapid, and reliable for the early screening of FHB resistance in wheat.

The barley UNICULM2 gene resides in a centromeric region and may be associated with signaling and stress responses.

Vegetative axillary meristem (AXM) activity results in the production of branches. In barley (Hordeum vulgare L.), vegetative AXM develop in the crown and give rise to modified branches, referred to as tillers. Mutations in the barley low-tillering mutant uniculm2 block vegetative AXM development and prevent tiller development. The objectives of this work were to examine gene expression in wild-type and cul2 mutant plants, fine map the CUL2 gene, and to examine synteny in the CUL2 region in barley with rice. RNA profiling experiments using two near-isogenic line pairs carrying either the cul2 mutant allele or wild-type CUL2 allele in different genetic backgrounds detected 28 unique gene transcripts exhibiting similar patterns of differential accumulation in both genetic backgrounds, indicating that we have identified key genes impacted by the CUL2 gene. Twenty-four genes had higher abundance in uniculm2 mutant tissues, and nearly half of the annotated genes likely function in stress-response or signal transduction pathways. Genetic mapping identified five co-segregating markers in 1,088 F2 individuals. These markers spanned the centromere region on chromosome 6H, and coincided with a 50-cM region on rice chromosome 2, indicating that it may be difficult to positionally clone CUL2. Taken together, the results revealed stress response and signal transduction pathways that are associated with the CUL2 gene, isolating CUL2 via positional cloning approaches that may be difficult, and the remnants of barley-rice synteny in the CUL2 region.

The genetics of barley low-tillering mutants: low number of tillers-1 (lnt1).

Barley (Hordeum vulgare L.) carrying recessive mutations at the Low number of tillers1 (Lnt1) gene does not develop secondary tillers and only develops one to four tillers by maturity. Double mutant analysis determined that the lnt1 mutant was epistatic to five of the six low and high tillering mutants tested. Double mutants of lnt1 and the low tillering mutant intermedium-b (int-b) resulted in a uniculm plant, indicating a synergistic interaction and that Lnt and Int-b function in separate tillering pathways. RNA profiling identified 70 transcripts with either increased or decreased abundance in the lnt1 mutant compared to wild-type. One gene with reduced transcript levels in the lnt1 mutant was the BELL-like homeodomain transcription factor JuBel2. The JuBel2 allele in the lnt1.a mutant contained a frameshift mutation that eliminated most of the predicted polypeptide, indicating that the Lnt1 gene encodes JuBel2. Previous studies with the low-tillering mutant absent lower laterals (als) showed that the tillering phenotypes and genetic interactions of als and lnt1 with other tillering mutants were very similar. However, the transcriptomes were very different; many transcripts annotated as stress and defense response exhibited increased abundance in the als mutant. This difference suggests a functional separation between Als and Lnt1 in the genetic control of tillering.

Addition of individual chromosomes of maize inbreds B73 and Mo17 to oat cultivars Starter and Sun II: maize chromosome retention, transmission, and plant phenotype.

Oat-maize addition (OMA) lines with one, or occasionally more, chromosomes of maize (Zea mays L., 2n = 2x = 20) added to an oat (Avena sativa L., 2n = 6x = 42) genomic background can be produced via embryo rescue from sexual crosses of oat x maize. Self-fertile disomic addition lines of different oat genotypes, mainly cultivar Starter, as recipient for maize chromosomes 1, 2, 3, 4, 5, 6, 7, 9, and the short arm of 10 and a monosomic addition line for chromosome 8, have been reported previously in which the sweet corn hybrid Seneca 60 served as the maize chromosome donor. Here we report the production and characterization of a series of new OMA lines with inbreds B73 and Mo17 as maize chromosome donors and with oat cultivars Starter and Sun II as maize chromosome recipients. Fertile disomic OMA lines were recovered for B73 chromosomes 1, 2, 4, 5, 6, 8, 9, and 10 and Mo17 chromosomes 2, 4, 5, 6, 8, and 10. These lines together with non-fertile (oat x maize) F(1) plants with chromosome 3 and chromosome 7 of Mo17 individually added to Starter oat provide DNA of additions to oat of all ten individual maize chromosomes between the two maize inbreds. The Mo17 chromosome 10 OMA line was the first fertile disomic OMA line obtained carrying a complete chromosome 10. The B73 OMA line for chromosome 1 and the B73 and Mo17 OMA lines for chromosome 8 represent disomic OMA lines with improved fertility and transmission of the addition chromosome compared to earlier Seneca 60 versions. Comparisons among the four oat-maize parental genotype combinations revealed varying parental effects and interactions on frequencies of embryo recovery, embryo germination, F(1) plantlets with maize chromosomes, the specific maize chromosomes retained and transmitted to F(2) progeny, and phenotypes of self-fertile disomic addition plants. As opposed to the previous use of a hybrid Seneca 60 maize stock as donor of the added maize chromosomes, the recovered B73 and Mo17 OMA lines provide predictable genotypes for use as tools in physical mapping of maize DNA sequences, including inter-genic sequences, by simple presence/absence assays. The recovered OMA lines represent unique materials for maize genome analysis, genetic, physiological, and morphological studies, and a possible means to transfer maize traits to oat. Descriptions of these materials can be found at http://agronomy.cfans.umn.edu/Maize_Genomics.html .

The genetics of barley low-tillering mutants: absent lower laterals (als).

Barley (Hordeum vulgare L.) carrying the recessive mutation absent lower laterals (als) exhibits few tillers and irregular inflorescence development. To gain an increased understanding of the genetic control of tillering in barley, we conducted morphological, genetic, and transcriptome analysis of the als mutant. Axillary buds for primary tillers, but not for secondary tillers, developed in als plants. Double mutant combinations of als with one low-tillering and four high-tillering mutants resulted in a tillering phenotype similar to als, indicating that als was epistatic to these tillering genes. However, double mutant combinations of als with another low-tillering mutant, intermedium-b, reduced tiller numbers, indicating there were at least two genetic pathways regulating tillering in barley. Next, we used simple sequence repeat markers to map the Als gene on the long arm of barley chromosome 3H, Bin 11. Finally, the Affymetrix Barley1 GeneChip was used to identify differentially accumulated transcripts in als compared to wild-type. Forty percent of the transcripts with twofold or greater accumulation in als tissues corresponded to stress and defense response genes. This finding suggested that a tillering pathway may modulate the stress response.

Maize centromere mapping: a comparison of physical and genetic strategies.

Centromere positions on 7 maize chromosomes were compared on the basis of data from 4 to 6 mapping techniques per chromosome. Centromere positions were first located relative to molecular markers by means of radiation hybrid lines and centric fission lines recovered from oat-maize chromosome addition lines. These centromere positions were then compared with new data from centric fission lines recovered from maize plants, half-tetrad mapping, and fluorescence in situ hybridizations and to data from earlier studies. Surprisingly, the choice of mapping technique was not the critical determining factor. Instead, on 4 chromosomes, results from all techniques were consistent with a single centromere position. On chromosomes 1, 3, and 6, centromere positions were not consistent even in studies using the same technique. The conflicting centromere map positions on chromosomes 1, 3, and 6 could be explained by pericentric inversions or alternative centromere positions on these chromosomes.

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

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

Maize DNA-sequencing strategies and genome organization.

A large amount of repetitive DNA complicates the assembly of the maize genome sequence. Genome-filtration techniques, such as methylation-filtration and high-CoT separation, enrich gene sequences in genomic libraries. These methods may provide a low-cost alternative to whole-genome sequencing for maize and other complex genomes.